The locomotor activity of freely moving rats was increased by electrical stimulation of brainstem sites, including the pedunculopontine nucleus, a major component of the mesencephalic locomotor region (MLR), and sites located in the subthalamic locomotor region (SLR), which is in the area of the zona incerta (ZI) dorsomedial to the subthalamic nucleus. Injections to the MLR of glycine, an inhibitory transmitter of the spinal cord and brainstem, had no effect on locomotion, nor did strychnine sulfate, a glycine antagonist. Unilateral injections of the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), and kainic acid, a glutamate analogue, into the MLR produced an increase in locomotion not seen with glutamate, an excitatory amino acid, into the same area. A still greater response, having a later onset than NMDA but also a longer duration, was produced by administration of picrotoxin and bicuculline methiodide, GABA antagonists, to the MLR. Carbachol injections into the MLR produced two types of responses: either increased or decreased locomotion. Hypermotility resulted from microinjections of glutamate, and picrotoxin and bicuculline, into the ZI. The short latency, short duration response to glutamate resulted in a greater increase in locomotion than with picrotoxin or bicuculline when each was administered into the SLR. These results provide further evidence for the functional role of the MLR and SLR in the initiation of locomotor activity in the intact, freely behaving rat.