L-serine-O-phosphate (L-SOP) is the immediate precursor to L-serine in the serine synthesis pathway and is also an agonist at the Group III metabotropic glutamate receptors (mGluRs). L-SOP is produced by the enzyme phosphoserine aminotransferase (PSAT) and metabolized to L-serine by phosphoserine phosphatase (PSP). Using a novel analytical procedure, we show that L-SOP is present in rat whole brain, and that in transfected cells, it is substantially more potent than L-glutamate at the mGluR4 receptor subtype. Immunocytochemical analyses showed that the distributions of PSAT and PSP in the cerebral cortex, hippocampus, and cerebellum were similar in the rat and macaque monkey brain. In the rat hippocampus, cells within the subgranular zone were co-labeled with anti-PSP and anti-PSA-NCAM, a marker for neurogenic cells. In the cerebellar cortex, Purkinje neurons expressed relatively high levels of both enzymes while robust expression of PSAT was also observed in the Bergmann glia. L-SOP released from Purkinje neurons or Bergmann glia could activate mGluR4 present on parallel fiber terminals. The presence of l-SOP in brain, its high potency at mGluR4, together with the restricted distributions of the synthetic and metabolic enzymes, suggest that L-SOP might act activate Group III metabotropic glutamate receptors in the CNS.