Thirty gallbladders were studied in vitro; 5 had black pigment stones and 25 contained manifestations of excess cholesterol in bile. Of the 25, 14 had cholesterol stones, 7 had macroscopic cholesterolosis, and 4 had cholesterol crystals. There were no differences in basal active tension among these groups, but the force of spontaneous phasic contractions was reduced in gallbladders with cholesterol stones, cholesterolosis, and cholesterol crystals compared with specimens with pigment stones (p < 0.001). The forces developed in response to cholecystokinin-8 (10⁻¹⁰–10⁻⁶ M), acetylcholine (10⁻⁷–10⁻³ M), and potassium chloride (20–60 mM) were greater in strips from specimens with pigment stones than in strips from specimens with cholesterol stones or cholesterolosis (p < 0.001). In cholesterol stones and cholesterolosis specimens, relatively strong muscle strips had similar responses to 10⁻⁶ M cholecystokinin-8 in normal calcium (2.5 mM) and in the absence of extracellular calcium. Weaker muscle strips had a reduced response to cholecystokinin-8 in the absence of extracellular calcium (p < 0.01). It is concluded that muscle strips exposed to bile with excess cholesterol have a reduced contractility compared with muscle strips from specimens with pigment stones; this impaired contractility precedes gallstone formation, and results from muscle dysfunction.