To investigate whether female sex hormones and pregnancy induce increased gallbladder synthesis of prostaglandin I2 (PGI2) and prostaglandin E (PGE), we used an in vitro incubation chamber to quantitate the effects of progesterone, estrogen, pregnancy, and pregnancy plus a 2%-cholesterol diet on mucosal and serosal PGI2 and PGE production by the rabbit gallbladder. Neither the female sex hormones nor pregnancy alone caused a significant increase in PGI2 or PGE synthesis. The gallbladders of cholesterol-fed, pregnant rabbits demonstrated significant increases only in serosal synthesis of PGI2. This increased production was equivalent to that noted for gallbladders from nonpregnant rabbits fed a high-cholesterol diet. There were no increases in mucosal synthesis of PGE or of PGI2. Thus, neither elevated levels of progesterone or estrogen nor pregnancy is directly responsible for the increased PGI2 activity in the female gallbladder; conversely, this effect seems to be mediated by the increased biliary concentrations of cholesterol.