In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for I gG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 2 19 x lo3 molecules/cell) compared to viable Kc (785 * 110 x lo3 moleculeslcell). Mean antigen density of HLA-DR and CDla molecules on viable LC were 579 -c 82 x lo3 molecules/cell and 1600 2 133 x lo3 molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases. o 1996 Wiley-Liss, Inc.