The efficiency of nitrous oxide in an equimolar mixture with oxygen or in concentrations up to 70% is approved for short painful procedures. Evaluation of the vitamin B12 levels in anesthetic staff applying nitrous oxide showed reduced vitamin B12 plasma levels. This study examines the vitamin B12 status of medical staff working with nitrous oxide in a pediatric emergency department (ED). Medical staff of the ED at the University Children's Hospital Zurich participated. The vitamin B12 status was evaluated by measuring homocysteine, methylmalonic acid, vitamin B12, blood count, and theMTHFR C677T genotype. As a control group, medical personnel working in the “nitrous oxide–free” pediatric intensive care unit were recruited.