Quantitative nonradioactive methods to measure the human immunodeficiency virus (HIV) in individually infected cells are needed for the direct assessment of HIV infection, for the evaluation of antiviral chemotherapy and for testing the efficacy of vaccines. As a first step in accomplishing this goal, we built an argon ion laser-based computerized image cytofluorometry (ALCIC) system and determined this instrument's ability to quantitatively measure HIV nucleotides in infected lymphocytes. ALCIC consisted of a 43-mW argon ion laser connected to a Zeiss Universal microscope via a fiberoptic cable, a charged coupled-device video camera, a video frame grabber and array processor and a Micro Vax II computer using computer programs written in FORTRAN. HIV RNA and DNA were detected in infected CEM lymphocytes in culture by in situ hybridization using acetylaminofluorene (AAF)-labeled HIV-DNA probes, a rabbit anti-AAF antibody and a fluorescein-labeled goat anti-rabbit antibody. ALCIC measurements showed that 61% of the CEM cells were infected and that quantitative differences were distinguishable within this group. The levels of fluorescein isothiocyanate (FITC) fluorescence were sixfold or more greater than that observed with the same system using a 100-W mercury lamp for illumination; the improved intensity of the laser-based system is due to greater excitation intensity of the laser and the matching of the excitation spectrum to the peak wavelength of FITC. ALCIC has potential clinical value for determining the effect of antiviral agents on HIV infection and for assessing the susceptibility of different cell types to infection.