Abnormal biliary motility has been observed in humans with gallstones and in animal models; however, the mechanism by which these abnormalities occur remains unknown. In this study we investigated the relationship between cholesterol gallstone formation, changes in biliary motility, and rates of gallbladder prostaglandin synthesis in prairie dogs receiving a 0.34% cholesterol diet for two, four, or six weeks. Gallstones did not occur until four weeks, when the incidence was 14%; after six weeks the incidence was 64%. Gallbladder emptying increased slightly at two weeks before becoming significantly decreased at four and six weeks. In contrast, there was a near linear increase in basal cystic duct resistance which began by two weeks of cholesterol feeding, although sphincter of Oddi resistances remained normal throughout the period of study. The synthesis of prostaglandins E and F2 alpha by the gallbladder was also increased beginning at two weeks and rose to a plateau at four and six weeks. In view of the potent effects of prostaglandins on biliary smooth muscle, these findings suggest that prostaglandins may mediate early changes in gallbladder and cystic duct motility which ultimately result in impaired gallbladder emptying.