Binding sites on human serum albumin (HSA) for some typical anionic drugs (Site I- and Site II-bound drugs) have been thermodynamically characterized by flow microcalorimetry. The binding and the thermodynamic parameters were computed directly from the calorimetric titration data at 37 degrees C in a phosphate buffer (pH 7.4) using one- and two-class binding models. From compensation analysis by plotting the molar enthalpy change (Delta H-m,H-i) versus the molar free energy change (Delta G,(m,i)), and the molar entropy change (Delta S-m,S-i) for every class of HSA binding sites, drug binding was classified into groups S1, S2 and S3, which agreed with those of fatty acid binding. Groups S1 and S2 included high-affinity binding sites (the first class of binding sites) and low-affinity binding sites (the second class of binding sites) for Site II-bound drugs, respectively, and group S3 contained the high-affinity binding sites for Site I-bound drugs. In each group, the Delta H-m,H-i-Delta S-m,S-i plot gave an excellent linear relationship where the value of T/P calculated from the intercept interpreted the molecular recognition by HSA as a quantitative measure of the hydrophobic interaction upon complex formation. Groups S1 and S2 were characterized by large negative values of Delta H-m,H-i and Delta S-m,S-i reflecting van der Waals interaction and hydrogen bonding formation in low dielectric media. The main force stabilizing the binding complex in group S3 was hydrophobic interaction characterized by small negative Delta H-m,H-i values, minor or positive Delta S-m,S-i values, and a large positive value of T Delta S-0 (32.4 kJ mol(-1)).