The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.