BACKGROUND: Although clinical management of drug resistance is routinely based on genotypic methods, phenotypic assays remain necessary for the characterization of novel HIV-1 inhibitors, particularly against common drug-resistant variants. We describe the development and assessment of the performance of a recombinant virus assay for measuring HIV-1 susceptibility to protease (PR), reverse transcriptase (RT), and integrase (IN) inhibitors. METHODS: The system is based on the creation of replication-competent chimeric viruses through homologous recombination between patient or laboratory virus-derived PCR fragments and the corresponding NL4-3 vector where the whole Gag-PR, RT-RNaseH or IN coding regions has been deleted through inverse PCR. The susceptibility to nucleoside (NRTIs) and non-nucleoside (NNRTIs) RT inhibitors and to IN inhibitors (INIs) is calculated through a single-round infection assay in TZM-bl cells, while protease inhibitor (PI) activity is determined through a first round of infection in MT-2 cells followed by infection of TZM-bl cells with MT-2 supernatants. RESULTS: The assay showed excellent reproducibility and accuracy when testing PI, NRTI, NNRTI, and INI susceptibility of drug-resistant clones previously characterized through the reference pseudoparticle-based Phenosense assay. The coefficient of interassay variation in fold change (FC) resistance was 12.0%-24.3% when assaying seven drug/clones pairs in three runs. FC values calculated by the Phenosense and in-house for 20 drug/clones pairs were in good agreement, with mean±SD ratio of 1.14±0.33 and no cases differing by more than twofold. CONCLUSIONS: The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes.