Streptococcus group B (GBS) is usually carried asymptomatically in the vaginal tract of women and can be transferred to the newborn during parturition. Serum antibodies to the capsular polysaccharide (CPS) can prevent invasive diseases, whereas immunity acting at the mucosal surface may be more important to inhibit the mucosal colonization of GBS and thus the risk of infection for the newborn. We prepared different GBS type III CPS-protein conjugate vaccines and evaluated their systemic and mucosal immunogenicity in mice. GBS type III CPS was conjugated to tetanus toxoid (TT) or recombinant cholera toxin B subunit (rCTB) either directly or to rCTB indirectly via TT. The conjugation was performed by different methods: (1) CPS was coupled to TT with 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide (EDAC), using adipic acid dihydrazide (ADH) as a spacer; (2) CPS was conjugated with rCTB using reductive amination; or, (3) N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used to bind rCTB to the TT of the CPS-TT conjugate. Mice were immunized with these conjugates or purified CPS by subcutaneous (s.c.) and intranasal (i. n.) routes. Antibodies to GBS III in serum, lungs and vagina were measured with ELISA. All of the CPS-protein conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates, when administrated s.c., induced only IgG responses in serum, lung and vagina, while i.n. vaccination also elicited IgA responses in the lungs and vagina. The CPS-TT conjugate administrated i.n. induced a strong serum IgG, but only a weak mucosal IgA response, while the CPS-rCTB conjugate elicited high IgG as well as IgA antibodies in the lungs after i.n. immunization. GBS III CPS-TT conjugated with rCTB produced a strong systemic and local anti-CPSIII response after i.n. administration. Co-administration of CT as adjuvant enhanced the anti-CPS systemic and mucosal immune responses further after i.n. administration with the CPS conjugates. These findings indicate that: (i) i.n. immunization with GBS CPS-protein conjugates was more effective than s.c immunization for stimulating serum as well as mucosal immune responses; (ii) rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; and (iii) the experimental GBS type III CPS conjugates containing rCTB should be investigated as mucosal vaccine to prevent GBS infection in humans.