Using serology, virology, and molecular epidemiology, we investigated nosocomial transmission of cytomegalovirus (CMV) over a two-year period in two contrasting environments: a crowded, busy pediatric chronic care unit (337 patients, 43 nurses, and 76 therapists; average prevalence of CMV excretion in patients, 16%) and a small neonatal unit (293 patients and 69 nurses; average prevalence, 0.7%). In the chronic care unit no nurse or therapist acquired CMV, but two pairs of infants were infected with homologous strains of CMV, and patient-to-patient transmission was proven in one pair. In the neonatal unit no patients acquired CMV in the hospital, but two nurses seroconverted, with a nonoccupational source proven for one. Transmission from CMV-infected caretaker to patient did not occur in either environment. CMV was isolated from diapers as well as hands of patients and personnel but not from other environmental surfaces.