The activity of a gamma-substituted glutamate analogue, (2S, 4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) and (2S,4R)-4-methylglutamic acid at ionotropic glutamate receptors has been examined. Ligand binding studies were performed using [3H] AMPA binding to membranes expressing either homomeric recombinant GluR1, GluR2, GluR4 receptors, and [3H] kainate binding to GluR5 and GluR6 kainate receptors. LY339434 and (2S,4R)-4-methylglutamic acid showed selectivity in ligand binding studies for kainate receptors over AMPA receptors. Within the kainate class of glutamate receptors, LY339434 showed selectivity for GluR5 over GluR6 whereas (2S,4R)-4-methylglutamic acid showed high affinity for both GluR5 and GluR6 kainate receptors. Examination of the functional activity of LY339434 and (2S,4R)-4-methylglutamic acid showed that both compounds evoked inward currents in dorsal root ganglion neurons (DRG) with estimated EC50 values of 0.8 +/- 0.2 microM and 0.17 +/- 0.04 microM, respectively. In GluR5 expressing HEK 293 cells, LY339434 evoked inward currents with an estimated EC50 value of 2.5 +/- 0.9 microM but had little effect on GluR6 expressing cells at concentrations less than 100 microM. LY339434 was a weak AMPA receptor agonist (EC50 values > 300 microM) as determined by activity in acutely isolated cerebellar Purkinje neurons. LY339434 and (2S,4R)-4-methylglutamic acid had agonist activity at NMDA receptors studied in cultured hippocampal neurons with EC50s of 2.5 microM and 11.7 microM, respectively. These results indicate that both LY339434 and (2S,4R)-4-methyl glutamic acid may be useful pharmacological tools for the examination of kainate receptors.