Despite the fact that most human subjects synthesize about twice as much cholic acid as chenodeoxycholic acid, available evidence suggests that 7α-hydroxycholesterol, the first intermediate in the major pathway for bile acid synthesis, is converted about equally to these two bile acids. Synthesis through the main alternate pathway can not explain this discrepancy because 27-hydroxycholesterol, the first intermediate in that pathway, is converted preferentially to chenodeoxycholic acid. To examine the validity of these contradictory observations, we administered [24−14C]-cholic acid and [24−14C]-chenodeoxycholic acid together with [7β−3H]-7α-hydroxycholesterol on one occasion and [22, 23−3H]-27-hydroxycholesterol on a separate occasion to eight normal human subjects. Synthesis of the two primary bile acids was determined by means of standard isotope dilution kinetics of the carbon 14-specific activities of biliary bile acids. Conversion of [7β−3H]-7α-hydroxycholesterol and [22, 23−3H]-27-hydroxycholesterol to bile acid was calculated from the tritium/carbon 14 ratio in cholic and chenodeoxycholic acid. For synthesis, the mean ± SEM cholic/chenodeoxycholic ratio was 1.82 ± 0.26. For apparent conversion of [7β−3H]-7α-hydroxycholesterol to bile acid, the mean ± SEM cholic/chenodeoxycholic ratio was 1.02 ± 0.09, whereas for [22, 233H]-27-hydroxycholesterol, the mean ± SEM cholic/chenodeoxycholic ratio was 0.38 ± 0.03. These data imply that, on average, more than 40% of cholic acid in these subjects was synthesized through a pathway that bypassed initial 7α-hydroxylation. However, consideration of all potential candidates for such a pathway raises doubts that any of them contributes substantially to bile acid synthesis.