The potential role of digestive endoscopy as a mode for transmission of hepatitis C virus (HCV) is controversial. To evaluate the role of digestive endoscopy in transmitting HCV by comparing the incidence of HCV infection in a cohort of patients undergoing endoscopy and in a cohort of blood donors. Prospective cohort study. 3 endoscopic units and 2 blood banks in northwestern Italy. The potentially exposed cohort consisted of 9188 outpatients consecutively recruited from 3 endoscopic units. Of 9008 patients negative for antibody to HCV (anti-HCV), 8260 (92%) were retested for anti-HCV 6 months after endoscopy. The unexposed cohort consisted of 51,230 healthy, anti-HCV-negative persons who donated blood at 2 blood banks in the same area and during the same time period; 38,280 of them (75%) were tested again for anti-HCV 6 to 48 months after the first blood donation (95,317 person-years of observation). Differences in the anti-HCV seroconversion rate between the exposed cohort (patients undergoing endoscopy) and the unexposed cohort (blood donors). Seroconversion was evaluated by a third-generation enzyme immunoassay for anti-HCV; persons positive for anti-HCV were tested for HCV RNA by polymerase chain reaction. All 8260 persons undergoing endoscopy remained negative for anti-HCV 6 months after the procedure (risk per 1000 persons, 0 [95% CI, 0 to 0.465]); in particular, none of the 912 patients who underwent endoscopy with the same instrument previously used on HCV carriers showed anti-HCV seroconversion (risk per 1000 persons, 0 [CI, 0 to 4.195]). Four blood donors became positive for anti-HCV and HCV RNA (mean follow-up, 2.49 years; 0.042 case per 1000 person-years [CI, 0.011 to 0.107 case per 1000 person-years]); each had undergone minor surgery before the second test. In the endoscopy cohort, 8.3% of patients were lost to follow-up. These findings support the hypothesis that properly performed digestive endoscopy is not a major risk factor for the transmission of HCV.