The purpose of this study was to evaluate the hormonal control of pyloric sphincter function. Studies were performed on both pyloric circular muscle, in vitro, and the human pylorus, in vivo. Full dose-response curves to gastrin I, cholecystokinin, and secretin were constructed for the pyloric muscle of the opossum studied at its length of optimal tension development, Lo. Both cholecystokinin and secretin were potent agonists on the muscle but gastrin I gave no increase in muscle tension. The combination of cholecystokinin and secretin was additive at submaximal concentrations but potentiation of the maximal responses was not observed. Gastrin I produced a surmountable, competitive-like antagonism to the effect of cholecystokinin on the pyloric muscle. The octapeptide of cholecystokinin was a more potent agonist than the whole molecule of cholecystokinin on the pyloric muscle. In man, the pyloric pressure rose significantly during intravenous infusion of either cholecystokinin or secretin. The combination of maximal doses of both hormones did not show significant potentiation. Gastrin I did not significantly increase pyloric pressure but did antagonize the pyloric response to duodenal acidification. These studies suggest that: (a) Both secretin and cholecystokinin augment pyloric sphincter pressure while gastrin I is an antagonist inhibiting their effects. (b) The hormonal responses of pyloric sphincter circular muscle, in vitro, can be correlated with human sphincter function, in vivo.