Herpes simplex virus type 1 gL lacks a transmembrane domain but stably associates with membranes through its oligomerization with the integral membrane glycoprotein designated gH. The gH-gL oligomers are essential for virion infectivity and virus-induced cell fusion. Monoclonal and polyclonal antibodies were raised against HSV-1(KOS) gL as probes for antigenic structure and functional protein domains. Antigenic determinants recognized by these antibodies were found to be present on gL expressed by many, but not all, strains of HSV-1 and were not detected on gL expressed by HSV-2 strains. These antigenic determinants were localized to the C-terminal region of HSV-1 gL, where amino acid substitutions define at least two classes of HSV-1 gL and where the sequences of HSV-1 and HSV-2 gL diverge considerably. The antibodies were extremely effective at inhibiting virus-induced cell fusion, provided the virus strain expressed the relevant antigenic determinants, but failed to neutralize viral infectivity despite demonstrable binding to virions. These results define strain-dependent differences in the structure and antigenic conformation of HSV-1 forms of gL and suggest that the roles of gL in cell fusion and viral entry are different.