BACKGROUND: Variation in medical practice has identified opportunities for quality improvement in patient care. The degree of variation in the use of antibiotics in children's hospitals is unknown. METHODS: We conducted a retrospective cohort study of 556 692 consecutive pediatric inpatient discharges from 40 freestanding children's hospitals between January 1, 2008, and December 31, 2008. We used the Pediatric Health Information System to acquire data on antibiotic use and clinical diagnoses. RESULTS: Overall, 60% of the children received at least 1 antibiotic agent during their hospitalization, including >90% of patients who had surgery, underwent central venous catheter placement, had prolonged ventilation, or remained in the hospital for >14 days. Even after adjustment for both hospital- and patient-level demographic and clinical characteristics, antibiotic use varied substantially across hospitals, including both the proportion of children exposed to antibiotics (38%-72%) and the number of days children received antibiotics (368-601 antibiotic-days per 1000 patient-days). In general, hospitals that used more antibiotics also used a higher proportion of broad-spectrum antibiotics. CONCLUSIONS: Children's hospitals vary substantially in their use of antibiotics to a degree unexplained by patient- or hospital-level factors typically associated with the need for antibiotic therapy, which reveals an opportunity to improve the use of these drugs.