The present study was undertaken to examine the effect of the hormonal environment on immunization with an attenuated strain of herpes simplex virus type 2 (HSV-2 TK−) and subsequent protection against challenge. Ovariectomized mice were administered saline (S; control), estradiol (E2), progesterone (P4), or a combination of estradiol and progesterone (E+P) and immunized intravaginally (IVAG) with HSV-2 TK−. Three weeks later, the immunized mice were challenged IVAG with wild-type HSV-2. Mice that were immunized following E treatment were not protected, whereas complete protection against the challenge was seen in mice from the S- and P4-treated groups. In the P4-treated group, 15% of mice developed chronic pathology following TK− immunization. Interestingly, about 40% of the E+P-treated mice were also protected. Upon examination of viral shedding in the vaginal secretions, it was clear that protection against challenge was dependent on the ability of the TK− virus to cause productive genital infection under different hormonal conditions. In the protected mice (the S and P groups and part of the E+P group), induced vagina-associated lymphoid tissues composed of CD11c+ dendritic cells and CD3+ and CD4+ T cells were formed transiently in the vaginal lamina propria from day 2 to day 5 postchallenge. These aggregates were absent in the unprotected mice (the E group and part of the E+P group). Significant HSV-2-specific activation of lymphocytes was observed in the local draining lymph nodes of protected mice. This response was absent in the unprotected groups. High titers of gB-specific local immunoglobulin A (IgA) antibodies were present in the vaginal secretions of S- and P4-treated immunized mice following HSV-2 challenge. The S-treated group of mice also had high gB-specific IgG titers. These studies show that sex hormones modify the induction of protective immune responses following IVAG immunization.