Human (HCMV) and guinea pig (gpCMV) cytomegaloviruses share biological similarities and DNA sequence homologies. Therefore, human and guinea pig sera were tested for cross-reactive antibodies. In eight human sere (four HCMV-seropositive and four HCMV-seronegative), low levels of neutralizing activity against gpCMV were not associated with antibodies to HCMV. The convalescent sera of one of three humans infected with either HCMV Towne vaccine or wild-type HCMV developed a fourfold increase in neutralizing titers to gpCMV after infection, but none of seven guinea pigs immunized with either HCMV Towne or purified HCMV gB developed neutralizing activity against gpCMV. Guinea pigs immunized with gpCMV did not develop antibodies to HCMV or human gB. Neither gpCMV, HCMV Towne stain or purified HCMV gB induced cross-reactive antibodies against the heterologous virus as detected by enzyme immunoassay. Our results indicate that gpCMV and HCMV share a very limited number, if any, of cross-reactive neutralizing epitopes.