Child care workers are potentially at risk for occupational exposure to cytomegalovirus, the leading cause of congenital infection in the United States. Preschool children often shed cytomegalovirus and commonly transmit virus to peers and parents. Workers from 32 day care centers were enrolled and tested for serum antibody to cytomegalovirus; 318 (62.5%) were seropositive. By logistic regression analysis the only variables significantly (P less than 0.05) associated with seropositivity at enrollment were older age and nonwhite race, though contact with younger children (less than 2 years of age) attained a P value of 0.06. Follow-up sera were obtained at 6-month intervals from 82 initially seronegative workers; 19 seroconverted in a median interval of 14 months, a rate of 20%/year, approximately 10-fold higher than the expected rate. The only demographic or employment variable associated with seroconversion was contact with children younger than 3 years of age for at least 20 hours per week (P = 0.03). Day care center workers have a markedly increased risk for acquisition of cytomegalovirus; those who could become pregnant should be appropriately counseled regarding prevention and consequences of cytomegalovirus infection during pregnancy.