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BACKGROUND AND AIMS Outbreaks of infections transmitted by duodenoscopes have prompted changes to duodenoscope cleaning protocols. However, little is known about effectiveness of clinical cleaning practices. We present culture data after duodenoscope manufacturer-recommended high-level disinfection (HLD) and quarantine. METHODS Our institution adopted a combination of manufacturer-recommended cleaning with the CDC-recommended culture and quarantine in 2015. Duodenoscopes (Olympus models TJF-Q180V, TJF-160, PJF-160) underwent HLD according to the manufacturer's reprocessing manual protocols after use. Two culture specimens were then obtained using a sterile brush from the distal tip, including elevator mechanism, and by flushing sterile water through the working channel. Duodenoscopes were quarantined until cultures resulted. Positive cultures were defined as >10 colony-forming units (CFUs) of low-concern organisms, or any CFUs of high-concern organisms according to CDC recommendations. If either culture specimen was positive, the process was repeated until cultures were negative. Data were collected from December 2015 to July 2018. RESULTS There were 140 instances of duodenoscope cleaning with 280 specimens. Twenty-eight of 234 (12%) initial culture specimens (18% of cultured duodenoscopes) were positive. Five of 36 (14%) second cultures were positive. Two of 8 (25%) third cultures were positive. Ninety-eight percent of organisms cultured were gram positive. There were 8 instances when both culture methods (brushing and flush) were positive; otherwise only one method was positive. There were 11 instances (8%) of duodenoscope removals from quarantine before final culture results. No patients had infections related to ERCP. CONCLUSIONS 18% of duodenoscopes had a positive culture after initial HLD. Only 3% were gram negative bacteria. Repeated HLD was 86% and 75% effective at eliminating initial and repeat positive cultures respectively. Initial HLD per manufacturer recommendations is not always effective at eliminating bacterial contamination. Additional steps are necessary to decrease risks of duodenoscope-transmitted infections.