Herpes simplex virus type 1 glycoprotein M (gM) is a type III membrane protein conserved throughout the family Herpesviridae. However, despite this conservation, gM is classed as a non-essential protein in most alphaherpesviruses. Previous data have suggested that gM is involved in secondary envelopment, although how gM functions in this process is unknown. Using transfection-based assays, we have previously shown that gM is able to mediate the internalization and subcellular targeting of other viral envelope proteins, suggesting a possible role for gM in localizing herpesvirus envelope proteins to sites of secondary envelopment. To investigate the role of gM in infected cells, we have now analysed viral envelope protein localization and virion incorporation in cells infected with a gM-deletion virus or its revertant. In the absence of gM expression, we observed a substantial inhibition of glycoprotein H-L (gH-L) internalization from the surface of infected cells. Although deletion of gM does not affect expression of gH and gL, virions assembled in the absence of gM demonstrated significantly reduced levels of gH-L, correlating with defects of the gM-negative virus in entry and cell-to-cell spread. These data suggest an important role of gM in mediating the specific internalization and efficient targeting of gH-L to sites of secondary envelopment in infected cells.