Successful vaccination against the human cytomegalovirus (HCMV) requires induction of both neutralizing antibody and cytotoxic T lymphocyte (CTL) responses. The HCMV glycoprotein B (gB, UL55) would be one of the most important immunogens to induce neutralizing antibodies. We tested the immunogenicity of an ALVAC (canarypox)-HCMV-gB (ALVAC-gB) recombinant in mice and guinea pigs in order to provide preclinical data for a phase I clinical trial of a HCMV vaccine candidate. ALVAC is an attenuated vaccine strain of canarypox virus which replicates productively in avian species but abortively in mammalian cells. The ALVAC-gB recombinant inoculated subcutaneously in mice and intramuscularly in guinea pigs induced HCMV-specific neutralizing antibodies and gB-specific CTL responses. Ultraviolet irradiation of the ALVAC-gB recombinant before immunization diminished CTL responses, indicating that intracellular expression and processing of gB-protein were necessary for CTL induction. Prior immunity to vaccinia virus did not decrease immunogenicity of the ALVAC-gB recombinant in mice. Thus, despite its host range restriction, ALVAC-gB is potentially capable of inducing both humoral and cell-mediated immune responses to HCMV in both vaccinia-immune and non-immune individuals.