Binding sites on human serum albumin (HSA) for anionic drugs and fatty acids have been thermodynamically characterized by microcalorimetry. The binding and the thermodynamic parameters were directly computed from the calorimetric titration data at 37 "C in a phosphate buffer (pH 7.4) using one-and two-class binding models. From compensation analyses plotting the molar enthalpy change (AHmj) versus those of the molar free energy (AGm,,) and molar entropy (ASmj) for each class of binding sites, HSA binding sites were classified into groups S1, S2, and S3. Group S1 included high-affinity binding sites for site Il-bound drugs, such as ibuprofen, flufenamic acid, and ethacrynic acid, and short-or medium-length alkyl-chain fatty acids; group S2 included low-aff inity binding sites of site Il-bound drugs and long-length alkyl-chain fatty acids; and group S3 contained the high-affinity binding sites for site I-bound drugs, such as phenylbutazone, oxphenbutazone, and warfarin, and long-length alkyl-chain fatty acids. High-and low-affinity bindings sites for salicylic acid and acetylaslicylic acid agreed with the regions of groups S3 and S2, respectively. Groups S1 and S2 were characterized by large negative values of AH,,,,i and AS, , , , , reflecting van der Waals interaction and hydrogen-bonding formation in low dielectric media, and the main force to stabilize the binding complex in group S3 was a hydrophobic interaction, characterized by a small negative AHmj and minor or positive values of A$,/ (entropy-driven).