Gallbladder bile from 24 patients (five with anaerobic biliary tract infection, 10 with aerobic biliary tract infection, and nine without biliary tract infection) was examined by thin-layer chromatography (TLC) for the presence of free bile acids. All five patients with an anaerobic biliary tract infection had small amounts of free bile acids in gallbladder bile. Seven of 10 patients with aerobic biliary tract infection and nine patients with sterile bile had no detectable free bile acids. Minute amounts of free bile acids were detectable on TLC in bile of three patients with aerobic infections. Calcium bilirubinate stones were more common in patients with free bile acids in bile.