A study was carried out to investigate an ATP-sensitive Ca2+ pool in rat epididymal cells and its role in transepithelial Cl- secretion. In normal buffered solution containing 2.5 mM free Ca2+, ATP triggered single calcium spikes in a dose-dependent fashion. In nominally Ca(2+)-free solution, the peaks of successive Ca2+ spikes diminished after repeated ATP stimulations. Addition of Sr2+ (2.5 mM) to Ca(2+)-free solution after ATP stimulation did not cause changes in fluorescence signals. However, in the presence of Sr2+, ATP gave rise to apparent repetitive Ca2+ spikes of similar magnitudes after repeated stimulations. Increasing the time of exposure in Ca(2+)-free solution containing 50 microM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid rapidly decreased the intracellular Ca2+ concentration ([Ca2+]i) response to subsequent ATP stimulation. On the other hand, increasing the time of exposure in Sr(2+)-containing solution in Ca(2+)-depleted cells rapidly increased the apparent [Ca2+]i response to subsequent ATP stimulation. These observations suggested the existence of a Ca2+ pool that was rapidly exchanging with the extracellular compartment. Apical application of ATP elicited a transient rise in short-circuit current across the epididymal epithelium in a dose-dependent fashion, and the response was reduced by prior stimulation with thapsigargin. Ca2+ released from a rapidly exchanging ATP-sensitive store might stimulate Cl- secretion in the epididymis, thereby maintaining the electrolyte contents and fluidity of the epididymal microenvironment.