The purpose of this study was to determine the nature of the neurochemical signals which impinge on the mesencephalic locomotor region (MLR) to produce locomotion in the rat. Injections of GABA antagonists into NADPH diaphorase-positive regions (PPN) were found to induce locomotion for short episodes (5-30 sec) which were repeated for several minutes (1-40 min). Such activity was blocked by injections of GABA and the GABA agonist, muscimol. Locomotion was induced by injection of substance P (SP), which also produced short, repeated episodes of locomotion. The more potent excitatory amino acid agonist, n-methyl-d-aspartic acid (NMDA), however, did produce dose-dependent, long-lasting (20 sec-5 min) locomotor episodes which were repeated over prolonged periods at the higher concentrations used (2-24 min). Additional injections of NMDA could drive stepping from a walk to a trot to a gallop. The effects of NMDA were blocked by injections of the excitatory amino acid antagonist, aminophosphonovalerionic acid (APV) (1-10 mM). Preliminary evidence suggests that carbachol (10-50 mM), a cholinergic agonist, inhibits NMDA-induced increases in muscle tone and episodes of stepping. The effect of carbachol was blocked by the cholinergic antagonist, atropine.