A flow cytometric assay has been developed to detect and quantitate human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells obtained from HIV-seropositive patients. Peripheral blood was obtained from patients attending an acquired immune deficiency syndrome clinic, and mononuclear cells were separated by centrifugation onto Ficoll-Hypaque. The cell layer at the interface was removed, washed in phosphate-buffered saline without Ca2+ and Mg2+, and fixed with 90% methanol, and intracellular HIV antigens were detected by indirect immunofluorescence with monoclonal antibodies to HIV antigens as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G F(ab')2 antibody as the secondary antibody. DNA content was determined by propidium diiodide staining after RNase treatment. These fluorochrome-treated cells were analyzed for two-color fluorescence by flow cytometry. The results showed that HIV-infected cells in peripheral blood that have been treated with monoclonal antibodies to the p24 or nef antigens of HIV can be detected and quantitated by flow cytometry. The percentage of p24 antigen-positive mononuclear cells had a significant correlation (P = 0.0001) with the clinical status of the patient, i.e., those with a high percentage of p24 antigen-positive cells had a poorer prognosis than those with a lower percentage of p24 antigen-positive mononuclear cells. In addition, for those in Centers for Disease Control groups III and IV, there was an inverse correlation between the percentage of p24 antigen-positive mononuclear cells and the number of T4 cells. However, cell-associated antigen detection by flow cytometry did not correlate with detection of antigen in sera of HIV-seropositive patients by the standard antigen capture enzyme-linked immunosorbent assay. This lack of correlation was probably due to the presence of immune complexes in the sera of HIV-seropositive patients. These results suggest that flow cytometry can be used as a rapid, sensitive, and quantitative assay system for the determination of the antigen status of HIV-seropositive patients and that it may be more useful as an indicator of disease progression than the currently used antigen detection methods.