We have examined the effect of L-glutamate and other excitatory amino acids on embryonic chick motoneurons maintained in cell culture along with other types of spinal cord cells. When the motoneuron membrane is clamped at -50 mV, glutamate induces a dose-dependent inward current. Although the dose-response curve is hyperbolic with an ED50 of 78 microM, glutamate apparently activates 2 types of receptors on motoneurons. The first, G1, is activated by N-methyl-D-aspartate (NMDA) and aspartate and inhibited by 2-amino-5-phosphonovaleric acid (2-APV). The second, G2, is activated by kainate and quisqualate and is not inhibited by 2-APV. At -50 mV, 38% of the glutamate current is due to activation of G1 receptors and the remaining 62% to G2 activation. In contrast to motoneurons grown with other spinal cord cells, sorted motoneurons grown in isolation apparently exhibit only G2 receptor-mediated currents. Both G1 and G2 currents reverse polarity between -10 and -5 mV. However, they could be distinguished when the membrane was hyperpolarized. G2 currents increased but G1 currents decreased when the membrane potential was increased beyond -50 mV. Consistent with the mixed agonist action of glutamate, glutamate currents remained nearly constant on hyperpolarization. No evidence was obtained that the G2 class of receptors on motoneurons could be subdivided: Quisqualate and kainate apparently compete for the same sites; gamma-glutamylglycine blocked quisqualate as effectively as it blocked kainate currents when the different potencies of the 2 agonists were taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)