Vaccines capable of protecting against sexually transmitted infections, such as human immunodeficiency virus (HIV), will depend on the induction of potent long-lasting mucosal immune responses in the genital tract. We evaluated vaginal and systemic immune responses and protection from vaginal challenge elicited after intranasal immunization of mice with inactivated glycoprotien 120-depleted HIV-1 immunogen alone or in combination with immunostimulatory CpG oligodeoxynucleotides (ODNs). Mice immunized with HIV-1 immunogen plus CpG ODN had significantly enhanced levels of anti-protein 24 immunoglobulin (Ig) G and IgA antibodies in serum and vaginal washes and increased production of beta-chemokines and interferon-gamma, compared with mice immunized with HIV-1 immunogen alone or with control ODN. Furthermore, mice intranasally immunized with HIV-1 immunogen plus CpG were protected against intravaginal challenge with a recombinant vaccinia virus expressing HIV-1 gag. These results indicate that mucosal immunization with whole-killed HIV-1 plus CpG ODN may be an effective means of inducing local immunity and protection against genital infection.