The patch-clamp method has been used to investigate the action of caffeine on the calcium current (Ic,) in single isolated smooth muscle cells of the guineapig ileum. Caffeine (10 mM) substantially inhibited Ic,. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K + in the pipette solution, application of caffeine evoked a transient Ca-dependent K + current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased Ic,. Ryanodine had only little effect on /Ca, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of /ca. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the Ic, inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of/ca inhibition was probably due to a direct blocking action of caffeine on Ca channels.