A murine model of genital infection with a thymidine kinase-deficient (tk⁻) strain of herpes simplex virus type 2 (HSV-2) was utilized to examine the local urogenital antibody response to HSV-2. Vaginal inoculation with HSV-2 tk⁻ protected against a subsequent genital challenge with a lethal dose of virulent HSV-2. After primary vaginal infection, predominantly HSV-specific IgG antibodies were detected in serum and vaginal secretions. HSV-specific IgG antibody-secreting cells (ASC) were detected first and in greatest numbers in the genital lymph nodes (gLN) after primary HSV-2 tk⁻ inoculation. HSV specific IgG or IgA ASC were not detected in the urogenital mucosa after primary HSV-2 vaginal infection. Vaginal HSV-2 challenge of HSV-immune mice resulted in increased HSV-specific serum IgG antibody and vaginal IgA antibody titers. HSV-specific IgG ASC were detected by 4 days postchallenge in gLN and by Days 6 and 7 postchallenge in the spleen and genital mucosa. These results suggest that urogenital humoral responses originate in the gLN following HSV genital infection and that gLN may serve as the primary source of the HSV-specific IgG- and IgA-secreting cells present in the urogenital mucosa after vaginal challenge.