A recombinant adenovirus (Ad) expressing glycoprotein B (gB) of herpes simplex virus (HSV) type 1 (AdgB8) was evaluated as a mucosal vaccine candidate. When administered intranasally (inl) to CS7Bl/6 mice, AdgB8 induced levels of serum anti-HSV gB IgG antibodies similar to those of mice immunized intraperitoneally (ip), which neutralized both HSV-l and -2. Mice immunized inl with AdgB8 produced secretory 19A specific for HSV gB, but mice immunized ip did not. Splenic anti-HSV cytotoxic T lymphocytes (CTL) were observed after inl and ip immunization; however, there was a time-dependent decrease in the anti-HSV CTL activity from spleens of inl immunized mice. Anti-HSV CTL were also present in the mediastinal lymph nodes after inl but not ip AdgB8 immunization. Furthermore, mice immunized inl with AdgB8 were protected against heterologous inl challenge with HSV-2, and this protection lasted longer than in ip-immunized mice. These results indicate that mucosal immunization with a recombinant adenovirus can induce mucosal and systemic immune responses and provide long-term protection from mucosally or sexually transmitted viruses.