The influence of deoxycholic acid (DCA) on the factors in gallbladder bile responsible for cholesterol gallstone disease has been a controversial subject of discussion. This might be partially due to patient selection or inappropriate methods. Therefore, we investigated the relationship between the percentage of DCA and lithogenic factors in the gallbladder bile of patients with cholesterol gallstones and with normal or moderately impaired gallbladder contractility. Patients with pigment stones served as a control group. The percentage of DCA in the gallbladder bile of 20 patients with cholesterol stones (23.2% ± 6.5%; mean ± SD) was comparable to the DCA percentage in the gallbladder bile of 11 patients with pigment stones (26.5% ± 8.5%). No correlation was seen between the DCA percentage of total bile acids and the crystal observation time, cholesterol saturation index (CSI), total protein value, mucin level, and amount of cholesterol in vesicles or crystals in the total group of patients or in the subgroups with cholesterol or pigment stones, respectively. The lack of correlation between DCA percentage and CSI was determined in native bile (r = 0.048) as well as in crystal-free bile after ultracentrifugation (r = 0.107). Our findings demonstrate that in patients with gallstones, the percentage of DCA in gallbladder bile is not related to any of the known biliary factors associated with cholesterol gallstone disease. We conclude that in patients with normal or moderately impaired gallbladder function, an elevated DCA level in the gallbladder bile is of minor pathophysiologic significance for the formation of cholesterol gallstones.