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Oral flora, blood-borne pathogens, and bacterial contamination pose a direct risk of infection to patients and health care workers. We conducted a study in a simulated operating room using a newly validated technology to determine whether the use of 2 sets of gloves, with the outer set removed immediately after endotracheal intubation, may reduce this risk.Forty-one anesthesiology residents (PGY 2–4) were enrolled in a study consisting of individual or group simulation sessions. On entry to the simulated operating room, the residents were asked to perform an anesthetic induction and tracheal intubation timed to approximately 6 minutes; they were unaware of the study design. Of the 22 simulation sessions, 11 were conducted with the intubating resident wearing single gloves, and 11 with the intubating resident using double gloves with the outer pair removed after verified intubation. Before the start of the scenario, we coated the lips and inside of the mouth of the mannequin with a fluorescent marking gel as a surrogate pathogen. After the simulation, an observer examined 40 different sites using a handheld ultraviolet light in the operating room to determine the transfer of surrogate pathogens to the patient and the patient’s environment. Residents who wore double gloves were instructed by a confederate nurse to remove the outer set immediately after completion of the intubation. Forty sites of potential intraoperative pathogen spread were identified and assigned a score.The difference in the rate of contamination between anesthesiology residents who wore single gloves versus those with double gloves was clinically and statistically significant. The number of sites that were contaminated in the operating room when the intubating resident wore single gloves was 20.3 ± 1.4 (mean ± SE); the number of contaminated sites when residents wore double gloves was 5.0 ± 0.7 (P < 0.001).The results of this study suggest that when an anesthesiologist wears 2 sets of gloves during laryngoscopy and intubation and then removes the outer set immediately after intubation, the contamination of the intraoperative environment is dramatically reduced.