Sulfated and non-sulfated bile acids were determined in the intestines and in the feces of 7-month-old germ-free and conventional male mice. 1. The bile acid pools in the gall bladder and small intestine were 21.13 mg/100g body weight in germ-free and 11.50 mg in conventional mice. The bile acid pools in the cecum and large intestine of germ-free mice were 3.03 mg/100 g body weight as compared to 1.24 mg in conventional mice. Fecal bile acid excretion was 2.93 mg and 4.12 mg/100 g body weight in 24 h in germ-free and conventional mice respectively. 2. The major bile acids from germ-free mice were cholic acid, alpha-muricholic acid and beta-muricholic acid. Small amounts of chenodeoxycholic and allocholic acid were also present. In addition to these primary bile acids the following secondary bile acids were identified in conventional mice: lithocholic, deoxycholic and omega-muricholic acid. 3. In both germ-free and conventional animals significant amounts of chenodeoxycholic and cholic acid were present as the 7-monosulfate esters. The sulfate esters of these bile acids did not exceed 2% of the total bile acids in the small intestine, but accounted for approximately 50% of the bile acids in the cecum and the large intestine. In contrast, the muricholic acids were nearly exclusively found in the non-sulfate fraction. 4. Alkaline hydrolysis without prior solvolysis of the sulfate esters resulted in loss of bile acids and production of artifacts. Hence, the bile acids of the mouse cannot be analysed by methods involving alkaline deconjugation unless a solvolysis step is included in the procedure.