An increasing body of evidence has suggested a role of pyrimidine nucleotides not only in metabolic pathways in the developing nervous system but also in adult brain functions. There is still little known about the cellular sources of pyrimidine synthesis and the distribution of enzymes involved in the biosynthetic pathway of pyrimidines in the central nervous system (CNS) of mammals. Therefore, we investigated the activity of dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the pyrimidine de novo synthesis, by catalytic enzyme histochemistry in adult rat brain. Its distribution was confirmed by Western blot analysis of dissected brain regions and by immunohistochemical analysis of paraffin-embedded formalin fixed sections employing an affinity-purified highly specific antibody. Microscopic analysis of brain sections revealed the presence of enzymatically active DHODH in many CNS regions, albeit at different intensities. High levels of both DHODH activity and immunoreactivity were observed in the neocortex, hippocampus, spinal cord and choroid plexus; lower levels were seen in the cerebellum, and only marginal expression in brainstem. The prominent staining of neuronal cell bodies in these regions suggests a neuronal location of DHODH. Neuronally derived pyrimidine de novo synthesis in the rodent CNS, independent of exogeneous sources, would provide pyrimidines for dynamic processes of membrane assembly, rearrangement and neuronal plasticity, as well as supplying uridine nucleotides as neuronal signalling molecules.