To study fetal brain infection with human cytomegalovirus (HCMV), an in vitro model was established using the human primary nontransformed neuronal cell line HCN-1A. On exposure to a mixture of factors promoting differentiation, HCN-1A cells differentiate into mature neurons. Both undifferentiated and differentiated neurons were permissive to HCMV replication as assessed by immunohistochemistry and in situ DNA hybridization. Infectious center assays revealed that the ratio of virus-infected differentiated cells to undifferentiated cells dropped from 11:1 to 2:1 7-21 days after infection. However, release of infectious progeny from the differentiated HCN-1A cells was greater by 100- to 1000-fold. Cytopathic effect appeared earlier and was more pronounced in differentiated cells. These results suggest that differentiation of HCN-1A cells dramatically affects the rate and amount of virus production from these cells. This model should be useful in the study of congenital HCMV disease and virus-host cell interaction.