A study was performed to determine whether differences in gallbladder proteins might be present in patients with rapidly nucleating bile. Gallbladder and hepatic bile protein concentrations were measured using a fluorometric assay. The method was validated by an independent technique, i.e., hydrolysis and amino acid analysis. Persons with cholesterol gallstones had significantly higher gallbladder bile protein concentrations than patients without gallbladder disease or patients with pigment stones. The protein concentration correlated with the in vitro nucleation time in the cholesterol stone group. Gallbladder bile proteins were also purified by chromatography and gradient ultracentri f ugation. Proteins from patients with cholesterol gallstones accelerated the nucleation time of control bile, whereas protein from controls had little effect. Hepatic bile protein concentrations were similar in persons with and without cholesterol gallstones. The gallbladderto-hepatic bile ratios of a variety of solutes were examined. The ratio for protein in the cholesterol gallstone group can be explained straightforwardly by water reabsorption in the gallbladder, whereas the very low ratio in patients without -cholesterol gallstones suggests that their gallbladders reduce protein mass by a process such as protein absorption or degradation during water absorption in the gallbladder.