OBJECTIVE: To determine whether contaminated ultrasound gel is the source of intermittent outbreaks of nosocomial infection due to Burkholderia cepacia complex in patients without cystic fibrosis since 1992. DESIGN: A prospective clinical and in vitro study of all in-use bottles of ultrasound gel, as well as a retrospective analysis of archived bacterial strains, were performed. Handling of gel for clinical purposes throughout the hospital was evaluated. Gel and archived clinical isolates of B. cepacia complex were speciated to genomovar level and characterized by pulsed-field gel electrophoresis, and the pulsed-field gel electrophoresis patterns were compared. SETTING: The Hospital for Sick Children, a 300-bed, tertiary care, pediatric academic health sciences center in Toronto, Canada. PATIENTS: All patients without cystic fibrosis from whom B. cepacia complex was recovered at the Hospital for Sick Children since 1992. RESULTS: No standardized protocol for storage or handling of ultrasound gel was found. Gel from 39% of bottles grew either B. cepacia (genomovar I) or Burkholderia stabilis (genomovar IV). These isolates had pulsed-field gel electrophoresis patterns identical to 2 of the 7 clinical pulsed-field gel electrophoresis types that are responsible for 88% of clinical isolates. CONCLUSIONS: Contaminated ultrasound gel contributed to nosocomial infection due to B. cepacia complex in this institution over the course of 10 years. Suggested guidelines for the handling of ultrasound gel are provided.