The structural basis of ligand specificity in human immunodeficiency virus (HIV) protease has been investigated by determining the crystal structures of three chimeric HIV proteases complexed with SB203386, a tripeptide analogue inhibitor. The chimeras are constructed by substituting amino acid residues in the HIV type 1 (HIV-1) protease sequence with the corresponding residues from HIV type 2 (HIV-2) in the region spanning residues 31-37 and in the active site cavity. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but has a decreased affinity for HIV-2 protease (Ki = 1280 nM). Crystallographic analysis reveals that substitution of residues 31-37 (30's loop) with those of HIV-2 protease renders the chimera similar to HIV-2 protease in both the inhibitor binding affinity and mode of binding (two inhibitor molecules per protease dimer). However, further substitution of active site residues 47 and 82 has a compensatory effect which restores the HIV-1-like inhibitor binding mode (one inhibitor molecule in the center of the protease active site) and partially restores the affinity. Comparison of the three chimeric protease structures with those of HIV-1 and SIV proteases complexed with the same inhibitor reveals structural changes in the flap regions and the 80's loops, as well as changes in the dimensions of the active site cavity. The study provides structural evidence of the role of the 30's loop in conferring inhibitor specificity in HIV proteases.