A simple quantitative bioassay for infectious HIV-1 has been developed. The assay is based on adherent CD4+ HeLa cell lines stably transfected with episomal vectors carrying theEscherichia coliβ-galactosidase gene under the control of the HIV long terminal repeat (LTR) promoter. HIV infection of these cell lines transactivates the LTR promoter inducing β-galactosidase production. Infected cells and virus foci can be stained dark blue by the addition of the chromogenic substrate X-Cal. Alternatively, a readily automatable quantitative enzyme assay can be performed on the infected cultures. Because of its simplicity the bioassay may be useful for routine quantification of HIV-infected cultures, plaque purification, virus neutralization studies and for the screening of antiviral agents.