This study examined the effect of hormonal environment on intranasal and subcutaneous routes of immunization in a genital herpes infection model. Ovariectomized mice were treated with estradiol (E(2)), progesterone (P(4)) or placebo hormone pellets and immunized intranasally (i.n.) or subcutaneously (s.c.) with attenuated HSV-2. Immunized mice were subsequently challenged, intravaginally, with wild-type HSV-2. Mice immunized under the influence of E(2) showed higher survival rates, reduced pathology and significantly lower viral shedding compared with those immunized under the influence of P(4) or placebo, by both i.n. and s.c. routes. Vaginal and serum anti-HSV-2 IgG, but not IgA, levels correlated with decreased pathology in E(2)-treated, i.n. immunized mice. We conclude that immunization under the influence of E(2) afforded better protection compared to placebo and P(4), by both routes of immunization. Female sex hormones can influence immune responses and outcome of viral challenge in the genital tract following systemic immunization.