OBJECTIVE A novel rapid reverse transcriptase (RT) recombinant HIV-1 drug-susceptibility assay was developed to evaluate resistance to RT inhibitors. METHODS HIV-1 RTs from five treatment-naive and 10 highly active antiretroviral therapy-experienced patients were evaluated. HIV-1 isolates recovered by culturing peripheral blood mononuclear cells from patients were used in the conventional isolate phenotype analysis. Recombinant HIV-1 strains were obtained by cloning the RT gene amplified from the supernatant of HIV-1 cultures in a plasmid carrying the HIV-1 strain HXB2 backbone, and the most represented clone for each virus isolate was then tested for antiviral drug susceptibility in parallel with HIV-1 isolates. RESULTS Comparison of conventional virus isolate and the novel recombinant virus phenotypic assays showed a large concordance of results. However, some discrepant results were observed, in that higher drug-resistance levels were detected by the conventional isolate phenotypic assay in HIV-1 isolates showing the presence of a mixture of HIV-1 variants, whereas the novel recombinant phenotypic assay could more precisely detect the level of drug resistance of the single viral clones selected for the analysis. CONCLUSIONS The novel recombinant phenotype assay, compared with the conventional virus isolate phenotype assay, showed widely overlapping results. The comparison of the two assays show that the conventional phenotypic assay is able to identify more efficiently the combined effect of drug-resistant viral variants, whereas the novel recombinant phenotypic assay is better able to define the level of drug resistance of the single viral variants. In addition, rapidity (2 weeks versus 4 weeks required by the reference recombinant assay and 6 weeks required by the conventional virus isolate phenotypic assay) is a major advantage of the novel assay.