Background. Nitrous oxide (N2O) inhibits methionine synthetase resulting in elevated plasma homocysteine (Hcy) concentration after surgery. In epidemiological studies, hyperhomocysteinaemia is associated with increased risk of cardiovascular disease and dementia.Methods. Blood samples were obtained to measure plasma folate and Hcy concentrations from two centres participating in a multicentre randomized trial investigating the effects of N2O on the outcome after major surgery. The effect of N2O and duration of anaesthesia on plasma Hcy, and the relationship between hyperhomocysteinaemia and outcomes were assessed.Results. We enrolled 394 patients. The N2O Group had an increase in plasma Hcy concentration after surgery when compared with the N2O-free Group: 11.1 (3.8) vs 8.5 (4.0) mu mol litre(-1), P < 0.0005. Postoperative hyperhomocysteinaemia was associated with an increased risk of major complications: risk ratio (RR) 2.8 (95% CI: 1.4-5.4), P=0.002 and cardiovascular events, RR 5.1 (95% CI: 3.1-8.5), P < 0.0005. There was a significant association between duration of anaesthesia and the relative change in plasma Hcy concentration, particularly in the N2O Group: r=0.42, P < 0.001.Conclusions. N2O increases plasma Hcy concentration; this effect is greater with a longer duration of anaesthesia. Hyperhomocysteinaemia is a risk factor for major postoperative complications. N2O-induced increases in plasma Hcy concentration may be a cause of postoperative cardiovascular morbidity.