We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.