The hepatic transport of five bile acids was studied in the bile-fistula rat. Pairs of labeled bile acids were injected simultaneously into the portal vein as a sharp pulse and the secretion of radiolabel in bile was measured over the nex 15 min. Six bile acid pairs were tested, and it was found that both conjugation and the number and disposition of hydroxyl groups influenced hepatic transport. Taurocholic acid was transported most efficiently, followed in order by glycocholic acid, cholic acid, deoxycholic acid, and chenodeoxycholic acid. Sampling aortic blood immediately after portal vein injection of labeled bile acid pairs demonstrated differential rates of hepatic extraction. In every comparison, the bile acid that was more efficiently extracted by the liver had a faster overall transport rate. This suggests that the differing rates of biliary secretion are, at least in part, determined by the efficiency of hepatic uptake.