Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.