Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors in rats. Specific activities of DHFR in cell cultures derived from human and rat primary intracranial gliomas and sarcomas were up to 6 times those found in adult rat liver. The presence of DHFR in neoplasms of central nervous system origin is relevant to the development of folate antagonists which, unlike methotrexate, can readily cross the blood-brain barrier. In normal developing rat brain, DHFR specific activity was high in embryos at 19 days of gestation and declined thereafter, until at 20 days after birth the activity was very low. The methotrexate titration assay was used to measure enzyme levels in the brains of fetal and newborn rats, and good correlation with the spectrophotometric assay was observed. The pattern was different in liver, showing maximum activity 11 days after birth and retaining high activity in adult liver. Both the cofactor requirement and the sensitivity to methotrexate indicate that the enzyme in the brain is DHFR.