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The kinetics of expression of the herpes simplex virus type 1-encoded major glycoprotein species gB, gC, gD, and gE on the surfaces of cells of murine, simian, and human origins were studied. Viable cells were stained with monoclonal antibodies specific for each species, and the levels expressed were determined by fluorescence flow cytometry. Differences were observed in both the kinetics and the levels of expression of individual glycoprotein species, depending upon the origin of the host cells. Glycoprotein gC was expressed early and at high levels in cells of murine and human origins, but late and at relatively low levels in simian cells. In contrast, gE was expressed at high levels in simian cells, but was not detectable until late in the infectious cycle in murine and human cells. The kinetics and levels of expression of gB were similar for all cells investigated, whereas gD, with high levels of expression in all cells late in infection, appeared on the surfaces of murine cells very early postinfection. This approach has allowed a simple quantitative method for comparing levels of glycoprotein expression.