Rapid and precise quantification of the infectivity of HIV is important for molecular virologic studies, as well as for measuring the activities of antiviral drugs and neutralizing antibodies. An indicator cell line, a CCD camera, and image-analysis software were used to quantify HIV infectivity. The cells of the P4R5 line, which express the receptors for HIV infection as well as β-galactosidase under the control of the HIV-1 long terminal repeat, were infected with HIV and then stained 2 days later with X-gal to turn the infected cells blue. Digital images of monolayers of the infected cells were captured using a high resolution CCD video camera and a macro video zoom lens. A software program was developed to process the images and to count the blue-stained foci of infection. The described method allows for the rapid quantification of the infected cells over a wide range of viral inocula with reproducibility, accuracy and at relatively low cost.