A methodology is presented for calculating relative binding free energies of enzyme-inhibitor associations in aqueous solvent. The methodology uses synthesis of semiempirical quantum chemistry to determine the protonation state of important residues in the enzyme active site, molecular mechanics to determine the gas-phase energetic contributions to the relative binding free energy, and dielectric continuum solvation to calculate electrostatic hydration contributions. The methodology is then applied to the calculation of the relative binding free energy of the inhibitors KNI-272, Ro31-8959, L-735,524, and A-77003 to HIV-1 protease and its I84V mutant. The calculated relative binding free energy is sensitive to the active-site protonation state of the aspartic acid residues of HIV-1 protease. The protonation state is inhibitor dependent. Given a particular protonation state, it was found that quantitatively accurate relative binding free energies could only be achieved when solvent effects were included. Three categories of binding were found. In the first, the change in binding free energy due to mutation is mainly due to the change in enthalpic interactions within the inhibitor-enzyme complex (Ro31-8959). In the second (L-735,524 and A-77003), the change in affinity is caused both by a change in enthalpic interactions within the enzyme and by a change in the hydration energy of the enzyme and inhibitor-enzyme complexes. In the third case (KNI-272), the change in affinity is mainly a solvent effect-it is due to changes in hydration of the enzyme only. In all cases, it was found that the I84V mutant enzyme was more stable than the wild-type enzyme. This alone (without consideration of the inhibitor-enzyme complexes) can qualitatively explain the reduction in binding affinity due to mutation.