N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a Cl- dependent, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) in vitro. To examine the pattern of NAALADase expression in the CNS, subcellular, regional, and developmental studies were conducted. Subcellular fractionation of lysed synaptosomal membranes revealed a substantial enrichment of the peptidase in synaptic plasma membranes as compared to mitochondrial or myelin subfractions. Regional studies reveal an apparent restriction of peptidase activity to kidney and brain. A threefold variation in specific activity was observed among brain regions, with highest specific activity in the cerebellum and lowest in telencephalic structures, a pattern that does not, in general, correlate with NAAG levels. Ontogenetic studies demonstrate a region-dependent, postnatal pattern of expression of NAALADase activity, with adult levels attained earliest in brainstem, as was previously reported for NAAG. Postnatal NAALADase expression would not appear to support a role for the peptidase in constitutive protein processing, but rather suggests that NAALADase may play a role in synaptic peptide degradation. Glutamate (Glu) excised from NAAG by NAALADase could be transported efficiently by uptake processes. Lesion studies, however, do not support a close structural association between NAALADase activity and the corticostriatal sodium-dependent, high-affinity, Glu uptake system. Similar to in vitro data documenting the route of NAAG degradation by NAALADase, after intrastriatal injection, NAAG was rapidly cleaved to two major products, N-acetyl-aspartate and Glu, with a t1/2 of approximately 10 min. Thus, the route of in vivo catabolism of NAAG parallels results from studies on NAALADase activity in vitro. These results are consistent with a role of NAALADase in the synaptic processing of NAAG. However, certain discrepancies in the regional and ontogenetic profiles of NAAG and NAALADase suggest that this relationship is not an exclusive one and may reflect a role for NAALADase on additional N-acetylated acidic peptides in vivo.