We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.