Ryanodine receptor type 2 (RyR2) is expressed in heart, smooth muscle and brain, and is considered to be the most general Ca²⁺-induced Ca²⁺ release (CICR) channel in sarco/endo-plasmic reticulum. We previously reported that, in excitation–contraction (E–C) coupling in smooth muscle cells (SMCs), elevation of [Ca²⁺] occurs via CICR in discrete local sites in subcellular spaces as Ca²⁺ hot spots and that the Ca²⁺ signal subsequently spreads to other parts of the cell to induce a contraction (Imaizumi et al. 1998; Ohi et al. 2001). It is not yet fully clear how much CICR via RyRs contributes to E–C coupling in SMCs. The present study on cells from humanely killed mice was undertaken to elucidate the significance of RyR2 in E–C coupling in urinary bladder (UB) SMCs using RyR2 heterozygous KO mice (RyR2^+/-), since homozygotes were embryonic lethal. RyR2 mRNA expression in RyR2^+/- was decreased by over 50% compared with wild-type mice (RyR2^+/+). RyR type3 mRNA expression was not changed in RyR2^+/-. The elevation of [Ca²⁺] in hot spots and whole-cell area at ~30 ms from the start of depolarization was significantly smaller in RyR2^+/- than in RyR2^+/+. Moreover, the number of hot spots was also smaller in RyR2^+/-. Contractile force development by electrical stimulation under moderate conditions was examined in the presence of atropine and some other antagonists of transmitter receptors and was also found to be smaller in RyR2^+/-. The force development was substantially decreased by 100 μM ryanodine in both but the extent of decrease was significantly smaller in RyR2^+/-, suggesting smaller a contribution of CICR to E–C coupling in RyR2^+/-. It has been well established that Ca²⁺ sparks activate BK channels to elicit spontaneous transient outward currents (STOCs), and STOCs regulate resting membrane potential and muscle tone (Heppner et al. 2003). The frequency of STOCs in single cell and the membrane depolarization by paxilline, a specific BK channel blocker, in tissue preparation were significantly reduced in RyR2^+/-. BK channel expression in RyR2^+/- was comparable to that in RyR2^+/+. These results strongly suggest that RyR2s play crucial roles in CICR for the regulation of E–C coupling and also in the generation of STOCs to regulate resting membrane potential and tone in UBSMC.