Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB- 11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10→Phe, Met-46→Ile, Ile-47→Val, and Ile- 50→Val. This is the first observation in HIV protease resistance studies of an Ile-50→Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50→Val mutant showed reduced sensitivity (two- to threefold) to VB- 11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46→Ile, Ile-47→Val, and Ile-50→Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50→Val displays a much lower affinity for the inhibitors than the parent enzyme (≤80-fold). The protease triply mutated at Met-46→Ile, Ile-47→Val, and Ile-50→Val shows an even greater decrease in inhibitor binding (≤270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L- 735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.