The role of B, CD4⁺ T and CD8⁺ T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated. Following primary inoculation with attenuated thymidine kinase-deficient (TK⁻) HSV-2, B cell-deficient (μMT) mice developed a local viraemia and transient genital inflammation, suggesting a role for B cells in the innate control of local infection and inflammation. Natural antibodies are implicated in this process, as passive transfer of normal serum into μMT mice significantly reduced HSV-2 TK⁻ shedding in the vaginal lumen, although it did not affect subsequent inflammation. Protection against lethal HSV-2 challenge was noted in HSV-2-vaccinated wild-type, CD8⁺ T cell-deficient and μMT mice and was characterized by strong virus-specific IFN-γ responses in vitro and delayed type hypersensitivity (DTH) responses in vivo. In contrast, CD4⁺ T cell-deficient (CD4^−/−) mice had impaired HSV-2-specific IFN-γ production and DTH responses and succumbed rapidly to genital HSV-2 challenge. However, protective responses to HSV-2 could be induced in HSV-2-vaccinated CD4^−/− mice by treatment with recombinant IFN-γ. Taken together, these results suggest that CD4⁺ T cells secreting IFN-γ are critical for immune protection against lethal genital HSV-2 re-infection, whereas B cells/natural antibodies have anti-viral and -inflammatory effects in the innate control of a primary infection.