Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25-50 nl 10(-2) or 10(-3) M D-[3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2-12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and gamma-D-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N-methyl-D-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.