To account for the wide variations in the prevalence of cytomegalovirus infections among day-care centers we serially tested 309 children at three day-care centers for 3 years. Based on the DNA restriction endonuclease pattern of each isolate, the rate of infection for children differ significantly (P < 0.001) among centers: at Center 1, 50% (46 of 93) of children acquired cytomegalovirus in day care; at Center 2, 62% (64 of 104); and at Center 3, 25% (21 of 84). Infection rates were associated with the number of infants enrolled, and half or more of infected children were younger than 24 months of age. Six of 7 new isolates were introduced by children 18 months of age. Based on DNA patterns the prevalent isolates at Centers 1 and 2, although different, were shed for an average of 22 and 23 months, respectively, compared with an average of 15 months for other isolates (P < 0.001). Reinfections with the prevalent isolates were observed for 2 of 34 children tested. The most important factors affecting day-care center transmission are the number of infants enrolled and prolonged viral shedding, possibly enhanced by reinfection.