Inhibitors of the human immunodeficiency virus (HIV) protease are a promising class of antiviral agents that dramatically reduce HIV replication both in culture and in infected patients. However, as for many other antiviral compounds, long-term efficacy of these agents is impeded by the emergence of virus variants with increased resistance to their inhibitory action, following selection of specific mutations in the protease coding sequence. We have studied HIV-1 variants that emerged at different stages of selection in the presence of the C2-symmetrical protease inhibitor ABT-77003. The selection of variants was a gradual process during which mutations accumulated at different sites in the protease, generating virus populations with increasing levels of resistance to the drug. The initially selected viruses had a low level of resistance as well as a markedly reduced replicative capacity. Further accumulation of mutations at secondary sites led to an improvement in both drug resistance and replication. In spite of their reduced infectivity, partially selected virus populations did not readily revert to wild-type when serially passaged in drug-free conditions. Instead, even in the absence of drug, secondary mutations identical to those selected in the presence of the inhibitor continued to emerge. These mutations improved both the intrinsic replicative capacity of the virus and its level of resistance to the inhibitor, suggesting that once committed to drug resistance, readaptation of the enzyme to its natural substrate leads to a reduction of its sensitivity to the inhibitor.