1. We used an isolated preparation of the embryonic chick brain stem and spinal cord to examine the origin, trajectory, and effects of descending supraspinal pathways on lumbosacral motor activity. The in vitro preparation remained viable for < or 24 h and was sufficiently stable for electrophysiological, pharmacological, and neuroanatomic examination. In this preparation, as in the isolated spinal cord, spontaneous episodes of both forelimb and hindlimb motor activity occur in the absence of phasic afferent input. Motor activity can also be evoked by brain stem electrical stimulation or modulated by the introduction of neurochemicals to the independently perfused brain stem. 2. At embryonic day (E)6, lumbosacral motor activity could be evoked by brain stem electrical stimulation. At E5, neither brain stem nor spinal cord stimulation evoked activity in the lumbosacral spinal cord, although motoneurons did express spontaneous activity. 3. Lesion and electrophysiological studies indicated that axons traveling in the ventral cord mediated the activation of lumbosacral networks by brain stem stimulation. 4. Partition of the preparation into three separately perfused baths, using a zero-Ca2+ middle bath that encompassed the cervical spinal cord, demonstrated that the brain stem activation of spinal networks could be mediated by long-axoned pathways connecting the brain stem and lumbosacral spinal cord. 5. Using retrograde tracing from the spinal cord combined with brain stem stimulation, we found that the brain stem regions from which spinal activity could be evoked lie in the embryonic reticular formation close to neurons that send long descending axons to the lumbosacral spinal cord. The cells giving rise to these descending pathways are found in the ventral pontine and medullary reticular formation, a region that is the source of reticulospinal neurons important for motor activity in adult vertebrates. 6. Electrical recordings from this region revealed that the activity of some brain stem neurons was synchronized with the electrical activity of lumbosacral motoneurons during evoked or spontaneous episodes of rhythmic motor activity. 7. Both brain stem and spinal cord activity could be modulated by selective application of the glutamate agonist N-methyl-D-aspartate to the brain stem, supporting the existence of functionally active descending projections from the brain stem to the spinal cord. It is not yet clear what role the brain stem activity carried by these pathways has in the genesis and development of spinal cord motor activity.