The proposal that folate polyglutamate cofactors of different chain lengths function differently in metabolism was investigated. We identified the one-carbon units present in rat liver folates within each of the liver folate polyglutamate groups, the folate penta-, hexa-, and heptaglutamates. This identification revealed that at the pentaglutamate level, 5-methyl-H4folate was the major form, accounting for 18% of the total liver folates, with small amounts of H4folate. At the hexaglutamate level, 5-methyl-H4folate and H4folate were major forms, accounting for 17% and 22% of the liver folates, respectively. At the heptaglutamate level, 5-methyl-H4folate occurred in small amounts while H4folate predominated. The rats used here had been fed a diet low in methionine and were exposed to N2O gas. These results are qualitatively similar to those from rat brain [Brody, T., Shin, Y. S., & Stokstad, E. L. R. (1976) J. Neurochem. 27, 409--413] where 5-methyl-H4folate was a major fraction of the folate pentaglutamates but a minor, if detectable, part of the hexa- and heptaglutamates. The folates 5-methyl-H4PteGlu5-7 were metabolically active in the liver, as illustrated by the severe contraction in the amounts of these folates following an injection of methionine. This indicates that "folate-binding proteins" do not prevent 5-methyl-H4folates from entering into one-carbon metabolism.