We determined the contribution of glutamate receptor subtypes to developing excitatory synaptic transmission in isolated spinal cord of rat embryos. Using electrophysiological and morphological techniques, we studied the pattern of development of synapses between dorsal root afferents and motoneurons in lumbar spinal cords of 15- to 21-d-old rat embryos. Motoneuron dendritic fields and afferent projections onto motoneurons were identified by labeling with HRP. Afferents first entered the gray matter at Day 15 of gestation, and by Day 16 they terminated close to motoneuron dendritic trees. Afferent axons projected onto motoneuron dendritic fields at Day 17, when boutons were detected on motoneuron dendrites that were crossed by afferent axons. To determine the time course of formation of functional sensorimotor synapses and their pharmacological properties, a dorsal root was stimulated while recording intracellularly from segmental motoneurons. At Day 16, excitatory postsynaptic potentials (EPSPs) with long latencies, slow rates of rise, and long durations were recorded. The amplitudes of these EPSPs increased with membrane depolarization and in the absence of extracellular Mg2+. These EPSPs were blocked by D-2- amino-5-phosphonovalerate (APV) and ketamine, which are selective antagonists of N-methyl-D-aspartate (NMDA) receptors. These findings suggest that initial synaptic transmission in embryonic motoneurons is mediated solely by NMDA receptors. Short-latency EPSPs with fast rates of rise were first recorded in most motoneurons by Day 17. These EPSPs were composed of fast- and slow-rising potentials. The slow component was blocked by APV, while the fast component was eliminated by 6-cyano- 7-nitroquinoxaline-2,3-dione and kynurenate. This indicates that the short-latency EPSPs are mediated by both NMDA and non-NMDA receptors. Dose-response curves of motoneurons to L-glutamate, NMDA, and kainate demonstrated that motoneurons are sensitive to these agonists prior to the formation of synapses between afferents and motoneurons. Motoneuron responses to NMDA and kainate increased immediately after the onset of short-latency EPSPs. This increased sensitivity could be due to extracellular factors influenced by growing sensory axons or intrinsic properties of differentiating motoneurons.