N-Acetylated alpha-linked acidic dipeptidase (NAALADase) is a neuropeptidase that may modulate glutamatergic neurotransmission. Independent of its characterization in the nervous system, one form of NAALADase was shown to be expressed at high levels in human prostatic adenocarcinomas, and it was designated the prostate-specific membrane antigen (PSMA). The NAALADase/PSMA gene is known to produce multiple mRNA splice forms, and based on previous immunohistochemical evidence, it had been assumed that the human brain and prostate expressed different isoforms of the enzyme. Because PSMA is being actively pursued as a target for autoimmune and cytotoxic targeting strategies to treat prostate cancer, the rigorous comparison of the two forms of the enzyme remained an important but untested question. To assess similarities and/or differences between human brain NAALADase and PSMA, we compared the two molecules using criteria of activity, immunoreactivity and sequences of the corresponding mRNAs. NAALADase from human cerebellar isolates displayed a kinetic profile and pharmacological sensitivities similar to PSMA. Also, Northern hybridization to PSMA cDNA detected indistinguishable sets of 2.8-, 4.0- and 6.0-kb RNA species in human brain and the LNCaP prostatic tumor cell line. In addition, the monoclonal antibody 7E11-C5 directed against the prostatic form of the enzyme immunoprecipitated 82% of human cerebellar NAALADase activity. Moreover, reverse transcription-polymerase chain reaction cloning of cerebellar cDNAs indicated that the human brain and prostate express a common mRNA splice form. Therefore, we conclude that the form of NAALADase also known as PSMA is expressed in brain and comprises a significant fraction of brain NAALADase activity.