Progestin-treated female mice are susceptible to vaginal infection by two sexually transmitted disease organisms: herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis. Vaccination of mice with HSV-2 or chlamydial antigens elicits immunity to vaginal infection that may be due in part to secreted antibodies in the vaginal lumen. Analysis of the role of these antibodies in immunity would be aided by information about the vaginal secretion in progestin-treated mice and the antibodies it contains. Gross and histologic observations of progestin-treated mice that were immune to vaginal HSV-2 infection indicated that the vaginal lumen was filled with mucus. A procedure for extraction of immunoglobulin from the mucus was developed and shown to recover at least 98% of the secretory IgA (S-IgA) that was free to diffuse from the mucus. Immunoblotting revealed that the predominant molecular form of IgA in vaginal mucus was dimeric S-IgA. Immunoglobulin concentrations in vaginal secretions were higher in immune mice than in non-immune mice and S-IgA concentrations were higher than those of IgG. The IgG concentration in vaginal secretions of immune mice was 4.5-fold higher than in non-immune mice, while serum IgG increased only 1.5-fold, suggesting local production of IgG or increased transudation in immune mice. Specific IgG antibody to HSV-2 was demonstrated in vaginal secretions of immune mice at a mean ELISA titer of 6200, whereas the titer of specific S-IgA in the same secretions was only 1.9. Thus, while the predominant immunoglobulin by weight in the vaginal mucus of immune mice was S-IgA, the ELISA titers suggested that the virus-specific antibody was almost entirely IgG.