Female guinea pigs were inoculated intravaginally with guinea pig cytomegalovirus (GPCMV) propagated either in guinea pig embryo fibroblast cultures (GPEF) or salivary glands. The incidence of infection was higher with GPEF virus. Rare instances of isolation of GPCMV from cervical swabs 9-48 hr after inoculation was attributed to survival of inoculum in the genital tract. Neither immunofluorescence microscopy nor histopathologic examination showed evidence for active infection of genital tract tissue examined up to day 5 after inoculation. At necropsy on days 30-49, GPCMV was isolated from salivary glands and occasionally from pancreas and lymph nodes. Seroconversion following intravaginal inoculation was demonstrated by an enzyme-linked immunosorbent assay (ELISA) test, and titers were comparable to those after intraperitoneal or subcutaneous inoculation. However, titers of neutralizing antibody, determined by plaque-reduction assay, were significantly lower in the group inoculated intravaginally. These findings are relevant to consideration of cytomegalovirus as a sexually transmitted agent in humans.