To examine the effect of changes in bile lithogenicity on gallbladder muscle function, in vitro gallbladder contractility was studied in an animal model of cholesterol gallstones: Richardson ground squirrels fed either a trace (control) or a 1% wt/wt cholesterol (test) diet. Lithogenic index of gallbladder bile increased on the test diet from 0.52 ± 0.03 to 0.81 ± 0.04 (p < 0.001). Isometric tensions generated in response to cholecystokinin-octapeptide, acetylcholine, or potassium depolarization, were all reduced > 50% in the test gallbladder muscles (p < 0.05), without any significant shift of the normalized dose-response curves. Tension in response to cholecystokinin-octapeptide differed significantly (p < 0.05) between each stage of stone formation compared with controls: 42% decrease in test animals before development of stones; 65% decrease in those with gallstones, ileal muscle from these animals, when tested with the same three stimuli, showed no adverse effects of the high-cholesterol diet. Another animal model, the prairie dog, also demonstrated a similar in vitro defect in gallbladder contractility associated with increases in bile lithogenicity. Thus, in the ground squirrel, a progressive defect in smooth muscle contractility to three different stimuli coincides with early changes in bile lithogenicity. The defect is not associated with any loss of sensitivity to these stimuli, and appears to be localized specifically to the gallbladder muscle, its presence in two animal models of cholelithiasis suggests that biliary stasis is an important factor in the early stages of cholesterol stone formation.