Abstract: The endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-α-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAA-LADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu-3H]NAAG were determined first (Km= 0.40 ± 0.05 μM; Vmax= 155 ± 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki= 1.9 ± 0.3 μM), and competitively by N-acetyl-L-aspartyl-β-linked L-glutamate (β-NAAG; Ki= 0.70 ± 0.05 μM). To determine whether glutamate-containing dipeptides, such as NAAG, β-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-γ-linked L-glutamate, were substrates of NAA-LADase, rat brain membranes were immobilized on a C-8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o-phthaldialdehyde/reverse-phase HPLC detection procedure. β-NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and β-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.