We examined the suitability of a TNF-beta cytokine ELISpot assay for assessing various aspects of the T cell response to herpes simplex viruses. The number of T cells responding to HSV-1 or HSV-2 was measured by TNF-beta ELISpot assay. The number of T cells producing TNF-beta in response to HSV-1 was high, ranging from 76 to 222 per 10(5). HSV-1-specific TNF-beta-secreting responder cell frequencies fluctuated over time in individual donors. Comparable fluctuations were not observed in the T cell frequencies to phytohemaglutinin (PHA). Responder cell frequencies to glycoproteins gB and gD of HSV-2 accounted for a large number of the HSV-2-specific T cells as measured using the TNF-beta ELISpot assay. Type-specific and type-common components of the T cell response to HSV-1 and HSV-2 could be estimated with this assay. Type-common responder cells typically accounted for 25-30%. Finally, CD4+ and CD8+ TNF-beta-producing T cells were stimulated by HSV-1 at a CD4:CD8 ratio of 2:1, indicating that both major subsets of T lymphocytes are activated by HSV.