The activation of phosphoinositide hydrolysis by ibotenate (IBO) in brain slices and the binding of N-[3H]acetylaspartyl-L-glutamate (NAAG) to brain membranes are biochemical parameters previously shown to be selectively inhibited by 2-amino-4-phosphonobutyrate (AP4). We have examined whether the binding of [3H]NAAG and stimulation of phosphoinositide hydrolysis by IBO are indexing the same or different populations of AP4-sensitive excitatory amino acid sites in brain. L-AP4 and D,L-2-amino-3-phosphonopropionate (D,L-AP3) were found to be about equipotent inhibitors of IBO-stimulated phosphoinositide hydrolysis. L-AP4 and D,L-AP3 did not inhibit stimulation of phosphoinositide hydrolysis by the cholinoceptor agonist carbachol. The L-isomers of serine-O-phosphate and alpha-aminoadipate were selective inhibitors of IBO-stimulated phosphoinositide hydrolysis, but were less potent than L-AP4 or D,L-AP3. When these compounds were examined for their ability to inhibit [3H]NAAG binding to membranes of rat forebrain, the relative order of potency was L-alpha-aminoadipate = D-alpha-aminoadipate greater than L-AP4 greater than L-serine-O-phosphate greater than D-AP4 much greater than D,L-AP3. Concentrations of NAAG up to 10(-2) M did not stimulate phosphoinositide hydrolysis. Thus, although both assays are sensitive to L-AP4 inhibition, they appear to represent disparate excitatory amino acid sites in brain. Furthermore, D,L-AP3 appears to be a more selective inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis than L-AP4, and might be a more useful pharmacological tool to define the function of these receptor sites in brain.