BACKGROUND Nitrous oxide inactivates vitamin B(12), inhibits methionine synthase, and consequently increases plasma total homocysteine (tHcy). Prolonged exposure to nitrous oxide can lead to neuropathy, spinal cord degeneration, and even death in children. We tested the hypothesis that nitrous oxide anesthesia causes a significant increase in plasma tHcy in children. METHODS Twenty-seven children (aged 10-18 years) undergoing elective major spine surgery were enrolled, and serial plasma samples from 0 to 96 hours after induction were obtained. The anesthetic regimen, including the use of nitrous oxide, was at the discretion of the anesthesiologist. Plasma tHcy was measured using standard enzymatic assays. RESULTS The median baseline plasma tHcy concentration was 5.1 μmol/L (3.9-8.0 μmol/L, interquartile range) and increased in all patients exposed to nitrous oxide (n = 26) by an average of +9.4 μmol/L (geometric mean; 95% confidence interval, 7.1-12.5 μmol/L) or +228% (mean; 95% confidence interval, 178%-279%). Plasma tHcy peaked between 6 and 8 hours after induction of anesthesia. One patient who did not receive nitrous oxide had no increase in plasma tHcy. Several patients experienced a severalfold increase in plasma tHcy (maximum +567%). The increase in plasma tHcy was strongly correlated with the duration and average concentration of nitrous oxide anesthesia (r = 0.80; P < 0.001). CONCLUSIONS Pediatric patients undergoing nitrous oxide anesthesia develop significantly increased plasma tHcy concentrations. The magnitude of this effect seems to be greater compared with adults; however, the clinical relevance is unknown.