N-Acetylaspartylglutamate (NAAG) is the most abundant neuropeptide in the mammalian nervous system. Considerable data support the hypothesis that NAAG is synaptically released in a manner consistent with neurotransmission. Primary murine brain cultures containing neurons and glia expressed 1.2-3.5 nmol of NAAG/mg of protein. In contrast to conclusions drawn from immunohistochemistry, pure glial cultures also expressed high levels of NAAG (0.6-2.11 nmol/mg of protein). These data suggest that although a subpopulation of neurons contains very high NAAG levels, micromolar concentrations of the peptide also are present in glia. Both culture types demonstrated robust extracellular peptidase activity when incubated with NAAG, as well as peptide transport. Uptake of [3H]NAAG was both temperature and sodium dependent, yet relatively insensitive to the presence of extracellular glutamate. These results indicate that synaptically released NAAG, as well as that which may be released from glia, is removed from the extracellular space by direct uptake as well as the robust enzymatic degradation of the peptide. A kinetic analysis of this NAAG transport (estimated Km = 1.8 microM) suggests a high-affinity NAAG transport system. The balance of the two processes of direct peptide uptake and peptide hydrolysis would markedly influence the sequence of receptor-mediated events that follow NAAG release.