Smooth muscle cells were isolated by collagenase digestion from the circular layer of the canine gastric corpus, and cell length was measured by image-splitting micrometry. Cells contracted following incubation with KCl, carbachol, and pentagastrin. While contraction induced by 40 mM KCl was inhibited by adding nitrendipine (10 nM) or by removing extracellular calcium and adding EGTA (2 mM), the contraction induced by carbachol (10 pM) was not affected by these measures. Nitrendipine partially inhibited contraction induced by pentagastrin, and pentagastrin also contracted cells that had been depolarized by prior exposure to KCl and nitrendipine. Removing extracellular calcium and adding EGTA partially inhibited contraction induced by maximally (10 pM) or submaximally effective (0.3 pM) concentrations of pentagastrin but did not affect that induced by 300 pM pentagastrin. These results indicate that isolated smooth muscle cells utilize calcium for contraction by three distinct mechanisms. The extent to which each mechanism contributes to contraction depends on the nature of the stimulus and, in the case of pentagastrin, the concentration of the stimulating ligand.