In contrast to intrauterine rubella infection, the relationship between timing of maternal cytomegalovirus (CMV) infection and fetal outcome has not been clearly defined. In order to investigate this relationship, a guinea pig model was utilized to assess the fetal consequences of maternal CMV infection during the first, second, or third trimester of pregnancy. Congenital infection occurred in 24 of 35 newborn guinea pigs (69%) delivered to mothers infected during the third trimester, with localization of virus to salivary gland in 17 of the 24 infected newborn guinea pigs. In contrast, only one of 28 (5%) progeny sacrificed following first-trimester maternal infection was congenitally infected (p less than 0.01). Second-trimester maternal infection was associated with an intermediate risk of intrauterine infection with transmission of virus to 17 of 54 progeny (33%) (p less than 0.01). Eight of the 10 fetuses delivered after second-trimester infection had virus in multiple organs including the brain. These data suggest that timing of maternal CMV infection is an important variable affecting fetal outcome, with increased risk of intrauterine infection when maternal infection occurs late in pregnancy. However, if fetal infection occurs earlier in pregnancy, it appears to present a greater threat to the fetus, with the potential for dissemination of virus in multiple fetal tissues, including the brain.