The neural control system for generation of locomotion is an important system for analysis of neural mechanisms underlying complex motor acts. In these studies, a novel experimental model using neonatal rat brain stem and spinal cord in vitro was developed for investigation of the locomotor system in mammals. The in vitro brain stem and spinal cord system was shown to retain functional circuitry for locomotor command generation, motor pattern generation, and sensorimotor integration. This system was exploited to investigate neurochemical mechanisms involved in neurogenesis of locomotion. Evidence was obtained for peptidergic and γ‐aminobutyric acid‐mediated mechanisms in brain‐stem circuits generating locomotor commands. Cholinergic, dopaminergic, and excitatory amino acid‐mediated mechanisms were shown to activate spinal cord circuits for locomotor pattern generation. EndogenousN‐methyl‐d‐aspartic acid receptors in spinal networks were found to play a central role in the generation of locomotion. The chemically induced patterns of motor activity and rhythmic membrane potential oscillations of spinal motoneurons were characteristic of those during locomotion in other mammals in vivo. The in vitro brain stem and spinal cord model provides a versatile and powerful experimental system with potentially broad application for investigation of diverse aspects of the neurobiology of mammalian motor control systems.— Smith, J. C.; Feldman, J. L.; Schmidt, B.J. Neural mechanisms generating locomotion studied in mammalian brain stem‐spinal cord in vitro.FASEB J.2: 2283‐2288; 1988.