The slow wave frequency of the canine colon has previously been hypothesized to be paced by an intracellular biochemical clock. We investigated the relationship between the endoplasmic reticulum (ER) Ca++ and the periodicity of the biochemical clock. Cyclopiazonic acid, a specific inhibitor of the ER Ca++ pump, dose-dependently decreased the pacemaker frequency. Similarly, chelating cytosolic Ca++ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) also decreased the pacemaker frequency. These observations suggest that delaying the Ca++ uptake into the ER decreases the pacemaker frequency. The pacemaker frequency was similarly decreased by neomycin [inhibiting inositol 1,4,5-triphosphate (IP3) synthesis] and by caffeine at concentrations higher than 5 mM (inhibiting the IP3-sensitive Ca++ channels in the ER membrane). Hence the IP3-sensitive Ca++ stores are involved in the biochemical clock. Ryanodine (up to 60 microM) did not affect the pacemaker frequency, which indicates that a ryanodine-sensitive store, if it exists, is not coupled to the biochemical clock. Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae that is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in interstitial cells of Cajal, slightly less in branching smooth muscle cells and far less in circular muscle cells. In summary, on the basis of these electrophysiological and morphological observations, we hypothesize that the Ca++ refilling cycle of the IP3-sensitive calcium stores associated with the plasma membrane determines the frequency of the pacemaker activity generated by the submuscular interstitial cells of Cajal-smooth-muscle network of the canine colon.