To show that an anti-human chorionic gonadotrophin-β (hCGβ) antibody response can be induced by inoculating Lb. Expressing hCGβ through diVerent mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 108, 109, and 1010 Lb.hCGβ (a recombinant Lactobacillus expressing hCGβ). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGβ IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGβ antigen-speciWc antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 109 and 1010 Lb.hCGβ inoculations induced similar anti-hCGβ antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 109 and 1010 Lb. hCGβ was able to neutralize more than 100 ng/ml hCGβantigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P <0.05). The numbers of anti-hCGβ IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGβ antibody levels in the reproductive tract.