BACKGROUND:: Nitrous oxide inactivates methionine synthase and may lead to DNA damage and wound infection. By using single-cell gel electrophoresis (comet assay), the authors determined the effect of nitrous oxide on DNA damage in circulating leukocytes. METHODS:: In this double-blind, randomized controlled trial, 91 patients undergoing major colorectal surgery were randomized to receive 70 nitrous oxide (n = 31) or nitrous oxide-free anesthesia using 30 (n = 30) or 80 (n = 30) oxygen. Venous blood was collected before and 24 h after surgery. The primary outcome was extent of DNA damage, quantified as the percentage of DNA staining intensity in the comet tail using digital fluorescence microscopy. Incidence of postoperative wound infection was also recorded. RESULTS:: Nitrous oxide exposure was associated with a two-fold increase in the percentage of DNA intensity in tail (P = 0.0003), but not in the 30 (P = 0.181) or 80 oxygen groups (P = 0.419). There was a positive correlation between the duration of nitrous oxide exposure and extent of DNA damage, r = 0.33, P = 0.029. However, no correlation was observed in nitrous oxide-free patients. The proportions of postoperative wound infection, using the Centers for Disease Control and Prevention criteria, were 19.4 (6 of 31) in the 70 nitrous oxide group and 6.7 (2 of 30) in both the 30 and 80 oxygen groups, P = 0.21. An increase in DNA damage was associated with a higher risk of wound infection, adjusted odds ratio (95 CIs): 1.19 (1.07-1.34), P = 0.003. CONCLUSIONS:: Nitrous oxide increased DNA damage compared with nitrous oxide-free anesthesia and was associated with postoperative wound infection. Copyright ? 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams Wilkins.