Acid-soluble pyrimidine nucleotides were separated by gradient elution ion-exchange chromatography after intracranial injection of L-carbamyl-C14-aspartate into mice. Radioactivity was associated with orotic acid, UMP, UDP, UTP, UDPAG, UDPG, CTP, CDP, CMP and CDP-ethanolamine; hence brain tissue is capable of synthesizing these pyrimidine nucleotides from simple precursors. Specific activities of the isolated nucleotides indicated that their biosynthesis by brain tissue occurs in a manner similar to that in rat liver and bacterial extracts. Comparison of specific activities of CTP, CDP and CMP suggests that uridine to cytidine nucleotide conversion takes place between UTP and CTP in mouse brain, probably by a reaction similar to that shown by others in extracts of E. coli. Distribution of radioactivity was the same in brains of mice previously infected with Theiler''s encephalomyelitis virus and in noninfected brains.