Interstitial cells of Cajal (ICs) are believed to play an important role by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. It was shown recently that single ICs from the rabbit portal vein (RPV) can generate rhythmical Ca²⁺ waves associated with membrane depolarisation and therefore may act as pacemakers in this tissue. Freshly isolated RPV ICs (freshly isolated from humanely killed rabbits) preloaded with the Ca²⁺ indicator fluo-3 and viewed by confocal microscope displayed rhythmical Ca²⁺ waves with a frequency from 1 to 6 waves/min. The frequency of waves correlated with the rate of rise of the fluorescence signal during the initial slow Ca²⁺ increase between Ca²⁺ waves, but not with their peak amplitude. This suggests that partial depletion of the endo/sarcoplasmic reticulum (E/SR) during each wave is not the factor determining the wave frequency and the initial slow Ca²⁺ increase is likely to reflect some pacemaking process, which modulates the wave frequency. Fast application of 5 mM caffeine during the declining phase of the slow Ca²⁺ wave evoked a Ca²⁺ transient of higher amplitude than the Ca²⁺ wave suggesting that intracellular stores are not completely depleted during Ca²⁺ wave generation. Ca²⁺ waves in the RPV were resistant to the L-type Ca²⁺ channel blocker nicardipine (10 μM) (n=10), but were abolished by Ca²⁺-free solution (n=2) and E/SR Ca²⁺-ATPase inhibitors, cyclopiazonic acid (10 μM) (n=4) or thapsigargin (1μM) (n=4). This suggests that Ca²⁺ release channels on the Ca²⁺ stores such as ryanodine receptors (RyRs) and IP₃ receptors (IP₃Rs) could be responsible for Ca²⁺ waves during slow wave generation. Blockers of the IP₃-induced Ca²⁺ release, 2-APB (30 μM) (n=6) and xestospongin C (10 μM) (n=4) abolished these waves suggesting involvement of IP₃Rs in the Ca²⁺ wave generation in RPV ICs. Application of ryanodine (100 μM) (n=5) abolished Ca²⁺ waves in the RPV ICs. Moreover, after blocking of Ca²⁺ waves with ryanodine, application of 5 mM caffeine (n=4) evoked a Ca²⁺ transient confirming that ryanodine directly inhibited the waves but not through depletion of the Ca²⁺ stores. Spatial distribution of S/ER and RyRs was visualised with a confocal z-sectioning protocol in ICs (n=20) freshly isolated from RPV loaded with the low affinity Ca²⁺ indicator fluo-3FF and stained with BODIPY TR-X ryanodine. A high degree of similarity between fluo-3FF and BODIPY TR-X ryanodine fluorescence suggested that all elements of intracellular calcium stores are enriched with RyRs. BODIPY TR-X ryanodine staining of ICs revealed RyRs irregularly distributed throughout the ICs with a continuous high-density region in the perinuclear E/SR. Simultaneous x-y confocal imaging of fluo-3 fluorescence revealed that slow Ca²⁺ waves originate locally within high-density RyR regions of the ICs.