Cholecystectomy has been hypothesized to cause increased levels of potentially harmful secondary bile acids due to prolonged exposure of primary bile acids to intestinal bacteria. In the present study, we analyzed duodenal bile of the same patients before and after cholecystectomy for keto bile acids and hydroxy bile acids. The ratios of individual keto bile acids to their corresponding precursor hydroxy bile acids were not significantly different before and after cholecystectomy. Keto bile acids constituted 2.5 ± 1.3 and 2.2 ± 0.9 mol% of hydroxy bile acids in duodenal bile. 3α-Hydroxy-12-oxo-5β-cholanoic acid derived from deoxycholic acid was the main keto bile acid in bile contributing 80 mol% to total keto bile acids. There was a strong positive correlation between 3α-hydroxy-12-oxo-5β-cholanoic acid and its precursor deoxycholic acid in bile (r = 0.88; p < 0.0005). Our findings prove that 7 weeks after cholecystectomy the percentage of keto bile acids in bile as compared to hydroxy bile acids is not incr