OBJECTIVE Apolipoprotein (apo) E is a genetically polymorphic protein influencing lipoprotein metabolism and the risk of both atherosclerosis and Alzheimer's disease. As opposed to common apo E3, apo E2 decreases and apo E4 increases hepatic lipoprotein uptake; hence, apo E4 could promote gallstone formation by increasing hepatic and biliary cholesterol concentrations. This study was designed to evaluate whether apo E polymorphism is related to gallstone risk. METHODS apo E phenotype was determined in subjects older than 40 years of age (160 with and 125 without gallstones) and in 61 patients with cholesterol gallstones who underwent cholecystectomy. Bile composition, nucleation time, and gallstone features were analyzed in surgical patients. RESULTS The E4/3 phenotype was enriched in both patients with gallstones and those who underwent cholecystectomy, with significantly (P < 0.006) higher epsilon 4 allele frequencies than in gallstone-free subjects (odds ratio, 2.67 [95% confidence limits, 1.23-5.93] and 3.62 [95% confidence limits, 1.49-8.91], respectively); women, but not men, accounted for these differences. The prevalence of the epsilon 4 allele increased with age in patients with gallstones, whereas the opposite occurred in gallstone-free subjects. Biliary lipid and gallstone cholesterol content tended to increase in the sequence E4 > E3 > E2 in patients who underwent cholecystectomy. CONCLUSIONS Carrying the apo E4 isoform is a genetic risk factor for cholelithiasis in humans, thus adding another adverse effect of apo E polymorphism on health.