We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4(+) cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 10(4) copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy.