Abstract: We have characterized a high-affinity [35S]-glutathione ([35S]GSH) binding site in mouse and human spinal cord. [35S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [35S]GSH binding was saturable, showing a Bmax of 72 fmol/mg of protein and a KD of 3.0 nM for mouse spinal cord and a Bmax of 52 fmol/mg of protein and a KD of 1.6 nM for human spinal cord. [35S]GSH binding was displaceable by GSH, l-cysteine, and S-hexyl-GSH, but not by glutamate, glycine, or NMDA. These [35S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [35S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [35S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [35S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.