The interaction of human serum albumin with twelve bile acids (ba) has been studied by equilibrium dialysis technique using 3H- and 14C-labeled bile acids. The physiological bile acids studied were: cholic, chenodeoxycholic, deoxycholic, lithocholic, ursodeoxycholic, and 7-ketolithocholic acids, all in the free and conjugated (with glycine and taurine) forms. For each bile acid studied, the interaction was characterized by two classes of binding sites, the first consisting of 2--4 sites and the second of 8--30. K1 values (liter/mol) for the different bile acids were: cholic acid, 0.3 x 10(4); chenodeoxycholic acid, 5.5 x 10(4); deoxycholic acid, 4.0 x 10(4); ursodeoxycholic acid, 3.8 x 10(4); 7-ketolithocholic acid, 1.9 x 10(4); lithocholic acid, 20 x 10(4). The affinity constant of a bile acid for albumin decreases with an increase in the number of hydroxy groups and also with the replacement of 7-hydroxy by 7-keto groups. The affinity constant is similar for glycine and taurine conjugated bile acids, but is slightly higher for unconjugated than conjugated forms.