Developmental characteristics of 5-methyltetrahydrofolate requiring N-methyltransferase (5 MT-NMT) have been studied in rats and mice and compared to the development pattern of marker enzymes. In rat and mouse brain, the 5 MT-NMT activity expressed in units per mg protein, was found to decrease rapidly during the first five days after birth and then more slowly, whereas two other enzymes, dopa-decarboxylase and histamine N-methyltransferase increased gradually during maturation. In kidney, the ontogenetic development of 5 MT-NMT was first characterized by an increasing activity from birth until a different age, depending on the species, and then followed by an abrupt decline. In contrast to this, histamine N-methyltransferase activity in mouse kidney was about 60 times higher at maturity than at birth. When the enzyme activities are expressed in units per g tissue, the changes in the course of the development of 5 MT-NMT were less apparent, but in any way differed markedly from those of dopa-decarboxylase and histamine N-methyltransferase. The results suggest that 5 MT-NMT does not behave as a synaptosomal or perhaps even as a neuronal enzyme. Although its physiological role remains to be elucidated, it must have a more general function in the cellular economy than merely N-methylating biogenic amines.