The ability of nitrous oxide (N2O) to disrupt folate and vitamin B12metabolism was examined in young (2-month), middle-aged (12-month), and elderly (24-month) Fischer 344 rats. Abnormalities in folate metabolism were assessed in a noninvasive manner by measuring the urinary excretion of formic acid and formiminoglutamic acid (FIGLU), compounds that are elevated in the urine of mammals with a deficiency in folate. After a 6-h exposure to 60% N2O/40% O2, urinary formic acid excretion increased 3− to 25-fold the first day following N2O exposure and returned to background levels by the second day after exposure in all age groups. Urinary FIGLU excretion increased 100− to 300-fold in the first day following N2O exposure, with the highest FIGLU excretion rates found in the elderly rats and the lowest in the young rats. By the second day after N2O exposure, FIGLU excretion rates returned to baseline levels in all age groups. Plasma folate progressively decreased with increasing age, whereas no age-dependent changes were observed in red cell folate, liver folate, or plasma vitamin B12levels. The elderly rats demonstrated the highest vitamin B12content in the liver and the lowest vitamin B12content in the kidney compared to the other age groups. Hepatic methionine synthase activities (measured 16–21 days after N2O exposure) were elevated in the elderly compared to the middle-aged or young rats, but methionine synthase activities in kidney and brain were not different among the three different age groups. It was concluded that in rats, agingper seonly slightly influences the disruption of folate metabolism produced by exposure to N2O.