To determine whether day-care workers acquire cytomegalovirus infection from the children they care for, we studied 610 women employed at 34 day-care centers over two years. Forty-one percent of the caretakers were seropositive for cytomegalovirus. After adjustment for the effects of race, marital status, and age on seropositivity, the women who cared for children younger than two years of age had a significantly higher seropositivity rate (46 percent) than the women who cared for children older than two years of age (35 percent) (relative risk, 1.29; 95 percent confidence interval, 1.05 to 1.57; P less than 0.02). Of 202 initially seronegative caretakers (observed for an average of 305 days per woman), 19 seroconverted, for an annual seroconversion rate of 11 percent. This rate was significantly higher than the 2 percent annual rate of seroconversion among 229 seronegative women (11 of whom seroconverted) in a comparison group of female hospital employees observed for an average of 781 days per woman (relative risk, 5.0; 95 percent confidence interval, 2.4 to 10.5; P less than 0.001). At three day-care centers in which the children were also studied, seven of the nine women shed isolates of cytomegalovirus in their saliva or urine that had EcoRI and BamHI DNA-digestion patterns identical to the DNA patterns of isolates shed by one or more children in their care. We conclude that workers in day-care centers may acquire cytomegalovirus infection from the children in their care and that this risk is significantly greater among those who care for children less than two years of age.