This study evaluated the effects of subunit guinea pig (GP) cytomegalovirus (CMV) immunization on congenital infection. Two 25-μg doses of an abundant GPCMV glycoprotein, the gp60-90 complex, plus adjuvant to GPs before pregnancy produced virus-specific humoral (neutralizing and ELISA) and cellular (proliferative and delayed type hypersensitivity) responses. Viral challenge before midgestation resulted in shorter maternal viremia in immunized than in unimmunized dams (3 vs. 17 days). Litters of immunized dams had reduced organ involvement and rates of congenital infection (48% vs. 18%) and increased mean birth weights (74 vs. 99 g). Amplification of DNA extracted from pup blood or organs with primers from the gB gene of GPCMV revealed congenital infection in some pups without detectable CMV by classic culture techniques. These data suggest that induction of preconception immunity to CMV by vaccine could be useful in reducing both the incidence and severity of congenital CMV disease. © 1995 The University of Chicago.