The actions of L-glutamate and agonists, agents blocking their membrane receptors and dorsal root afferent volleys, were compared on intracellularly recorded neuronal activity in an in vitro horizontal slice preparation of the hamster spinal dorsal horn. Bath-applied L-glutamate or L-aspartate (less than or equal to 1 mM) rapidly depolarized and excited less than a third of the dorsal horn neurons sampled. Bathing solutions containing low Ca2+ eliminated synaptic transmission in the slices but failed to block the excitatory effects of L-glutamate for the majority of the neurons tested. N-Acetylaspartylglutamate had no effect on dorsal horn neurons at concentrations up to 1 mM. Neurons excited by L-glutamate were most commonly located in the superficial dorsal horn (laminae I and II). Neurons insensitive to L-glutamate were more broadly distributed, with a number being located in laminae III-V. Kynurenic acid, 2-amino-4-phosphonobutyric acid, and 2,3-piperidine dicarboxylic acid selectively antagonized rapid, short-lasting synaptic components of the dorsal cord potentials. Kynurenic acid reversibly antagonized intracellularly recorded L-glutamate-induced excitation, spontaneous synaptic potentials, and fast synaptic potentials evoked by dorsal root volleys. Compounds with strong antagonist actions at the NMDA receptor, 2-amino-5-phosphonovaleric acid and D-alpha-aminoadipic acid, were much less effective in suppressing the effects of L-glutamate or in blocking synaptic potentials. We conclude that a subset of spinal neurons directly excited by dorsal root fibers have excitatory membrane receptors activated by L-glutamate. This conclusion is consistent with the concept that L-glutamate or a substance binding to the receptors it activates is released from the central terminals of some primary afferent fibers and mediates fast synaptic transmission from them to certain spinal neurons in the dorsal horn.