In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)-sensitive intracellular Ca2+ store and the effects of the Ca(2+)-induced Ca2+ release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 mumol/L) caused a transient release of Ca2+ from an intracellular store, presumably via an inositol tris-phosphate-sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca2+ but was obtained only once in Ca(2+)-free bathing solution, which shows that a depleted intracellular Ca2+ store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L), implying that Ca2+ enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 mumol/L) caused a large Ca2+ release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca2+]i that were dependent on extracellular Ca2+. Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca2+ transient but instead slowly increased [Ca2+]i. It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca2+ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)