In order to determine the cellular localization of gamma-glutamyl transpeptidase (gamma-GTP) in the nervous system the enzyme was purified to homogeneity and used to prepare an antiserum in rabbits. The anti-gamma-GTP serum cross-reacted with rodent tissues, but not those of human, monkey, calf, sheep or chicken. Monospecificity of the antibody was demonstrated by immunoelectrophoresis. The antibody inactivated the purified enzyme in a dose-dependent manner. The gamma-GTP antibody was used with the indirect fluorescent method to visualize sites containing gamma-GTP in the CNS. Choroid plexus, capillaries and the luminal side of ependymal cells stained well. Additionally, immunoreactive cells were found in both grey and white matter throughout the CNS which did not have the morphological appearance of neurons and were thought to be glia. These immunocytochemical studies suggest that gamma-GTP is largely though not exclusively associated with glia in the CNS.