Various permeabilization procedures and defined gating techniques were tested in order to optimize existing flow cytometric assays and devise a specific assay for the direct detection of intracellular HIV‐1 antigens in clinical blood specimens. In our optimal procedure, blood lysed with Orthomune Lysing Reagent was fixed with 3.7% formaldehyde for 10 min at room temperature and then permeabilized with 0.2% Tween 20 for 15 min at room, temperature. Cells from whole blood were labeled with either FITC‐anti‐p18 or FITC‐anti‐p24 monoclonal antibodies and PE‐anti‐Leu M9 (CD33) in order to exclude monocytes and granulocytes from the lymphocyte gate. The assay demonstrated that mean percentages of HIV p24 antigen positive cells were increased in patients with advanced disease. The assay in its present form is useful for monitoring disease progression and for monitoring the effects of antiviral therapy in individuals, but it is not currently sensitive enough to detect consistently the low levels of HIV infected peripheral blood cells in asymptomatic individuals. © 1994 Wiley‐Liss, Inc.