Possible interactions between different intracellular Ca2+ release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca2+ signals. Spontaneous, local Ca2+ transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 μm) but not by the inositol 1,4,5-trisphosphate receptor (IP3R) blocker, 2-aminoethoxydiphenyl borate (100 μm), identifying them as Ca2+ sparks. Caffeine (10 mm) and carbachol (10 μm) initiated Ca2+ release at sites which co-localized with each other and with any Ca2+ spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml−1) both inhibited the global cytoplasmic [Ca2+] transient evoked by carbachol, confirming that it was IP3R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca2+ store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP3Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca2+ release through co-operation between IP3Rs and RyRs, and implicate IP3Rs in the regulation of Ca2+ store content.