We prospectively studied day-care providers at six day-care centers in southeastern Iowa to determine their occupational risk for primary cytomegalovirus infection and to define epidemiologic risk factors. Ninety-six (38%) of 252 day-care providers were seropositive for cytomegalovirus by latex agglutination at entry into the study. Among 82 seronegative providers available for follow-up, seven seroconversions occurred at only two of the six participating centers, yielding an annualized seroconversion rate of 7.9%. Median time to seroconversion among these providers was 13 months. Using Kaplan-Meier estimates of risk, we determined that the overall risk of seroconversion among providers at various centers ranged from 0% to 22% by 12 months and from 0% to 40% by 16 months. Risk of cytomegalovirus acquisition by providers was independent of race, age, education, the presence of a child at home, or caring for children younger than 2 or 3 years in the day-care center. However, the risk of seroconversion among day-care providers appeared to parallel rates of cytomegalovirus excretion and acquisition among children at each center.