A flow cytometric assay (FCA), which detects the p24 antigen in HIV-infected cell lines and in peripheral blood mononuclear cells (PBMC) of AIDS patients, has been described in several studies. However, the results presented here clearly show that this p24-FCA, although useful for the analysis of HIV infection of cells in vitro, does not specifically detect HIV-infected PBMC from patients. Isotype control antibodies also stained PBMC from HIV-infected patients to a greater degree than the PBMC from healthy controls. Furthermore, the CD4-negative lymphocytes, which are generally not infected with HIV, were also found to stain with anti-p24. Finally, no enrichment of HIV-infected cells was found in the FACS-purified CD4+p24+ lymphocytes, compared to the CD4+p24- cell fraction. The p24-FCA, therefore, was not useful for determining the percentage of infected PBMCs from HIV-infected individuals.