Lesions resulting from recurrent genital herpes simplex virus (HSV) infection are characterized by infiltration of CD4+ lymphocytes. We have investigated the antigenic specificity of 47 HSV-specific CD4+ T-cell clones recovered from the HSV-2 buttock and thigh lesions of five patients. Clones with proliferative responses to recombinant truncated glycoprotein B (gB) or gD of HSV-2 or purified natural gC of HSV-2 comprised a minority of the total number of HSV-specific clones isolated from lesions. The gC2- and gD2-specific CD4+ clones had cytotoxic activity. The approximate locations of the HSV-2 genes encoding HSV-2 type-specific CD4+ antigens have been determined by using HSV-1 x HSV-2 intertypic recombinant virus and include the approximate map regions 0.30 to 0.46, 0.59 to 0.67, 0.67 to 0.73, and 0.82 to 1.0 units. The antigenic specificity of an HLA DQ2-restricted, HSV-2 type-specific T-cell clone was mapped to amino acids 425 to 444 of VP16 of HSV-2 by sequential use of an intertypic recombinant virus containing VP16 of HSV-2 in an HSV-1 background, recombinant VP16 fusion proteins, and synthetic peptides. Each of the remaining four patients also yielded at least one type-specific T-cell clone reactive with an HSV-2 epitope mapping to approximately 0.67 to 0.73 map units. The antigenic specificities of lesion-derived CD4+ T-cell clones are quite diverse and include at least 10 epitopes. Human T-cell clones reactive with gC and VP16 are reported here for the first time.