The vast majority of HIV-1 infections worldwide are caused by the C and A viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including those identified as targets for antiretroviral therapies. In the case of the HIV-1 protease, we reported earlier [Velazquez-Campoy et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6062-6067] that proteases from the C and A subtypes exhibit a higher biochemical fitness in the presence of widely prescribed protease inhibitors. In this paper we present a complete thermodynamic dissection of the differences between proteases from different subtypes and the effects of the V82F/I84V drug-resistant mutation within the framework of the B, C, and A subtypes. These studies involved four inhibitors in clinical use (indinavir, saquinavir, ritonavir, and nelfinavir) and a second-generation protease inhibitor (KNI-764). Naturally occurring amino acid polymorphisms found in proteases from the C and A subtypes lower the binding affinities of existing clinical inhibitors by factors ranging between 2 and 7.5 which by themselves are not enough to cause drug resistance. The preexisting lower affinity in the C and A subtypes, however, significantly amplifies the effects of the drug-resistant mutation. Relative to the wild-type B subtype protease, the V82F/I84V drug-resistant mutation within the C and A subtypes lowers the binding affinity of inhibitors by factors ranging between 40 and 3000. When the enzyme kinetic properties (k(cat) and K(m)) are included in the analysis, the biochemical fitness of the C and A subtype drug-resistant mutants can be up to 1000-fold higher than that of the wild-type B subtype protease in the presence of the studied inhibitors. From a thermodynamic standpoint, the combined effects of the drug-resistant mutations and the natural amino acid polymorphisms on the Gibbs energy are additive and involve significant alterations in the enthalpy and entropy changes associated with inhibitor binding. At the biochemical level, the combined effects of naturally existing polymorphisms and drug-resistant mutations might have important consequences on the long-term viability of current HIV-1 protease inhibitors.