Context: Vaccine development to prevent congenital cytomegalovirus (CMV) infection has been impeded by the uncertainty over whether maternal immunity protects the fetus. Objective: To determine whether the presence of maternal antibodies to CMV significantly reduces the risk of congenital CMV infection in future pregnancies. Design, Setting, and Participants: Cohort study of 3461 multiparous women from a population with a high rate of congenital CMV infection who delivered newborns screened for congenital CMV infection between 1993 and 1998, and whose cord serum specimen from a previous delivery could be retrieved and tested for antibody to CMV. Main Outcome Measure: Congenital CMV infection according to maternal immune status, age, race, parity, and socioeconomic status. Results: Of 604 newborns born to initially seronegative mothers, congenital CMV infection occurred in 18 (3.0%). In contrast, of 2857 newborns born to immune mothers, congenital CMV infection occurred in 29 (1.0%) Two factors, preconception maternal immunity (adjusted risk ratio, 0.31; 95% confidence interval, 0.17-0.58) and maternal age of 25 years or older (adjusted risk ratio, 0.19; 95% confidence interval, 0.07-0.49), were highly protective against congenital CMV infection. No other factors were associated with a reduction in the risk of congenital CMV infection. Conclusion: Naturally acquired immunity results in a 69% reduction in the risk of congenital CMV infection in future pregnancies.