The recombinant virus assay (RVA) is a method for assessing the susceptibility of human immunodeficiency virus type 1 (HIV-1) plasma isolates to antiretroviral drugs. The RVA involves the production of viable virus in vitro by homologous recombination of RT-PCR products from plasma virus with a noninfectious reverse transcriptase (RT) or protease (PR)-deleted cloned HIV-1 provirus. In this study, we have constructed RVA plasmids with contiguous deletions in RT, PR, and the p7/p1 and p1/6 gag protease cleavage sites (CS). The deletions in these plasmids allow generation of recombinant viruses with all loci currently identified as important for resistance to anti-HIV-1 drugs being derived from the clinical isolate, including CS mutations that compensate for the reduced fitness of viruses resistant to protease inhibitors (Doyon et al., J Virol 1996:70:3763-3769). We have also used these new constructs to generate viruses with or without compensatory CS mutations, and examined the effects on fitness. In the case of an indinavir-selected virus, fitness was restored close to that of a wild type virus when a vector deleted in the CS and PR was used. With an amprenavir-selected isolate, virus fitness was incompletely restored by including the CS, and this defect appeared to be partially due to reduced infectivity of the virions. We conclude that the CS mutations were required for optimum detection of resistance in the RVA, but that virus fitness can remain compromised even in the presence of compensatory CS mutations.