10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) is a folate-binding protein that is important in folate metabolism. In addition, 10-FTHFDH catalyzes the rate-limiting step in hepatic folate-dependent formate oxidation. We measured folate concentrations and examined cellular 10-FTHFDH expression in regions of the adult rat central nervous system (CNS), to study components of CNS oxidative formate metabolism. Folate was detected in every CNS region studied; the concentrations ranged from 3 to 14% of that detected in the liver. Immunohistochemical expression of 10-FTHFDH was identified in many CNS structures. 10-FTHFDH was mostly expressed by glia, especially astrocytes and ventricular ependyma. Neuronal expression was weak but evident in the cerebral cortex, basal ganglia, cerebellum, and spinal cord. Thus, CNS tissue has the chief components of folate-dependent formate oxidation and the chief site of this oxidation appears to be glia.