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Rotavirus is the most common etiologic agent of healthcare-acquired diarrhea in pediatric patients. There has been little published information on healthcare-acquired rotavirus infection.This was a retrospective cohort study of children hospitalized with rotavirus gastroenteritis at our institution between December 1999 and May 2004. Patients with community- and healthcare-acquired rotavirus gastroenteritis were compared with regards to age, time of infection, patient unit, and viral subtype as determined by reverse transcription polymerase chain reaction sequencing.Five hundred seventy-seven children were hospitalized with rotavirus gastroenteritis during the study period. One hundred twenty-one (21%) of these infections were healthcare-acquired. The incidence of healthcare-acquired infection was 4.2 cases per 10,000 patient-days. With the exception of 1 outbreak on an isolated patient unit, community- and healthcare-acquired disease affected similar patient populations, had the same temporal distribution, and were caused by viruses with similar subtypes. However, there was a significant difference between the geographic distribution of community- and healthcare-acquired disease within the hospital (P < 0.001). The majority (83%) of community-acquired cases were admitted to general medicine-surgery units, but only 53% of the healthcare-acquired cases occurred on these units (P = 0.005). The remaining healthcare-acquired infections occurred on units that rarely admitted patients with community-acquired disease.Healthcare-acquired rotavirus gastroenteritis seems to be caused by repeated introduction of community strains into the hospital setting. Heightened attention to infection control practices and rapid rotavirus identification is necessary on all units, especially those that infrequently admit children with rotavirus gastroenteritis, to prevent the spread of healthcare-acquired disease.