Objective To study the activity of aminopeptidase N (APN) in the bile of patients with cholesterol gallstones (CGS) and rabbits fed with lithogenic diets (LD) to explore the lithogenic role of APN in the formation of gallstones. Methods Enzymatic activity of APN and lipid concentrations in bile of investigated CGS patients and LD fed rabbits were measured. The relationship between the activity of APN and CSI was statistically analyzed.Results APN activity in CGS patients′ gallbladder (GB) and bile duct (BD) biles 〔(66 0?24 1)*!U/ml and (38 4?9 4)*!U/ml〕 as well as in LD fed rabbits′ GB and BD biles 〔(14 5?4 08)*!U/ml and (9 13?1 67)*!U/ml〕 were significantly higher than that in the corresponding biles of their clinical and experimental control (CC and EC) groups 〔(23 0?7 7)*!U/ml〕 in GB bile of CC group and 〔(6 05?0 95)*!U/ml and (3 09?0 92)*!U/ml〕 in EC group. All P values were less than 0 05. A statistical positive correlation was found between the APN and CSI value in each of the groups.Conclusions It is more likely that APN might play certain role in the early neucleating period of the pathogenesis of gallstones based on its proneucleative potential and measurement of APN activity may serve as a predictive marker among patients with high risk of gallstone formation.