The cellular mechanisms which determine the frequency of spontaneous activity were investigated in gastric smooth muscles isolated from the guinea-pig. Intact antral muscle generated slow waves periodically; the interval between slow waves was decreased exponentially by depolarization of the membrane to reach a steady interval value of about 7 s. Isolated circular muscle bundles produced slow potentials spontaneously or were evoked by depolarizing current stimuli. Evoked slow potentials appeared in an all-or-non fashion, with a refractory period of 3 ~ 5 s. Low concentrations of chemicals which modify intracellular signaling revealed that the refractory period was causally related with the activity of proteinkinase C (PKC). Activation of PKC increased and inhibition of PKC activity decreased the frequency of slow potentials. Chemicals which inhibit mitochondrial functions reduced the frequency of slow waves. Inhibition of internal Ca2+-store activity decreased the amplitude, but not the frequency of slow potentials, suggesting that the amplitude is causally related to Ca2+ release from the internal store. The results suggest that changes in [Ca2+]i caused by the activity of mitochondria may play a key role in determining the frequency of spontaneous activity in gastric pacemaker cells.