The significance of functional coupling between ryanodine receptor (RyR) and Ca²⁺-activated ion channels in Ca²⁺ spark sites has been well established in various types of smooth muscle cells (SMCs)(Wellman & Nelson, 2003). In contrast, experimental results about the functional significance of Ca²⁺-induced Ca²⁺ release (CICR) in excitation-contraction (E-C) coupling have been still controversial in SMCs (Collier et al. 2000; Herrera et al. 2000). We examined here i) the relative contribution of CICR versus direct Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) during single action potential (AP) to a contraction, ii) mechanisms underlying the spread of Ca²⁺ wave from local areas to whole cell, and iii) mechanisms of Ca²⁺ sequestration, during E-C coupling in single urinary bladder (UB) SMC of humanely killed mouse and guinea-pig. In addition to analyses of confocal Ca²⁺ images and membrane currents, following four approaches have been made in this series of studies. 1) CICR via RyR was completely blocked by 100 μM ryanodine. When UBSMCs were activated by 30 ms depolarization from –60 to 0 mV in the absence of ryanodine, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevated in several discrete small areas along cell membrane as hot spots in the early stage of depolarization (< 10 ms). Ca²⁺ hot spots, then, spread to whole cell area more slowly as Ca²⁺ waves even after repolarization (Ohi et al., 2001). A shorter depolarization (5 ms) elicited only a few Ca²⁺ sparks, which usually disappeared quickly. The number of Ca²⁺ hot spots elicited by depolarization closely related to the duration of depolarization up to ~20 ms. There was a sort of threshold duration around 10 ms to induce enough number of Ca²⁺ hot spots to spread globally as waves. Application of 100 μM ryanodine from a patch pipette changed neither the resting [Ca²⁺]_i nor the amplitude of VDCC current but abolished Ca²⁺ hot spots and markedly reduced peak [Ca²⁺]_i upon depolarization. Application of 3 μM xestospongin C, a blocker of inositol trisphosphate receptor (IP₃R) markedly reduced acetylcholine-induced Ca²⁺ release but did not affect Ca²⁺ hot spots and waves upon depolarization. Addition of 100 μM ryanodine to UB tissue segments reduced amplitude of contraction induced by direct electrical stimulation under moderate conditions by ~70 % in the presence of antagonists for transmitter receptors. 2) Ca²⁺ concentrations in cytosol ([Ca²⁺]_i) and mitochondria matrix ([Ca²⁺]_m) were monitored in a cell by fluo-4 and rhod-2, respectively. Upon depolarization, [Ca²⁺]_i was immediately elevated in hot spot sites and reached the peak at ~150 ms. In contrast, [Ca²⁺]_m was increased with a delay of ~50 ms after depolarization and peaked at ~500 ms. Upon repolarization, [Ca²⁺]_i returned to the resting level with a half decay time of ~500 ms, while [Ca²⁺]_m recovered more slowly (~1.5 s). The depolarization-induced increase in [Ca²⁺]_m in discrete mitochondria, where a Ca²⁺ hot spot was located closely, was significantly larger than that without the co-localization. 3) The contribution of VDCC to E-C coupling was examined in UBSMC of mutant mice, which lacked β3 subunit of VDCC (β3^-/-). In β3^-/- UBSMC, Ca²⁺ current density was reduced by 45 %. The mRNA expression of RyRs and BK channel in UBSM of β3^-/- was comparable to that in β3^+/+, respectively. The amplitude of contraction induced by moderate electrical stimulation in UB tissue segments of β3^-/- was comparable to that in β3^+/+ but high K⁺ induced contraction in β3^-/- was significantly smaller than that in β3^+/+. The treatment with 100 μM ryanodine reduced contraction by the electrical stimulation in larger extent in β3^-/- than in β3^+/+. Collectively, β3 knock-out resulted in reduction of VDCC density, which was functionally compensated by larger contribution of CICR to a twitch contraction. 4) The contribution of RyR type 2 (RyR2) to E–C coupling was examined in UBSMs from RyR2 heterozygous KO mice (RyR2^+/-), in which RyR2 mRNA expression decreased by over 50 % of wild type (RyR2^+/+). The number of Ca²⁺ hot spots and elevation of [Ca²⁺]_i upon depolarization were significantly smaller in RyR2^+/- than those in RyR2^+/+. The density of VDCC and BK channel currents was not changed in RyR2^+/-. The contraction by moderate electrical stimulation and its decrease by ryanodine were significantly smaller in RyR2^+/- than in RyR2^+/+. In conclusion, CICR during E-C coupling elicited by single AP occurs in two steps in UBSMC. Ca²⁺ influx does not increase [Ca²⁺]_i markedly but elicits initial CICR in discrete hot spot sites via functional coupling between VDCC and RyR2. In the second step, Ca²⁺ release spreads as waves to other Ca²⁺ stores with amplification but more slowly in a way without mediating activation of IP₃Rs. Two steps of CICR are essential for a twitch contraction elicited by single AP in UBSMC.