The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase, on the distribution of folic acid derivatives in rat bone marrow cells, kidney, brain, and liver were determined. Methionine synthetase activity was decreased by about 90% in bone marrow cells, kidney, and brain and by 83% in liver. The proportion of 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in N2O-exposed rats increased from 1.4- to 1.9-fold depending on the tissue examined. This increase was at the expense of a decrease in different folate derivatives in different tissues--in bone marrow cells, kidney, and liver 5-HCO-H4PteGlu, 10-HCO-H4PteGlu, and H4PteGlu decreased; in brain only H4PteGlu decreased significantly. Total endogenous folates, as measured by Lactobacillus casei after conjugase treatment, were unchanged in all tissues after nitrous oxide exposure. The results are interpreted as direct support of the methyl trap hypothesis as the explanation of the interrelationship of folate and vitamin B12 metabolism in bone marrow cells, kidney, and brain, as well as in liver.