During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.