Experiments were designed to examine the effect of chronic pretreatment with progesterone (2 mg/kg . day for 5 days) on the in vitro contractile response of gallbladder muscle strips from adult male guinea pigs. The muscle strips were challenged with either acetylcholine or the octapeptide of cholecystokinin and dose-response relationships were determined. The data were compared with dose-response curves obtained from untreated control animals. progesterone pretreatment produced right-ward shifts in the acetylcholine and octapeptide of cholecystokinin curves that were characterized by significant decreases in the maximal contractile response. Serum progesterone concentrations in the pretreated animals were significantly increased over control levels. The data support the hypothesis that the sluggish behavior of the gallbladder during pregnancy and during the luteal phase of the menstrual cycle may be due, in part, to progesterone-related alterations in the contractile properties of gallbladder smooth muscle.