Abstract In this report we describe the development of an in vitro preparation of the mudpuppy ( Necturus maculatus) used to investigate locomotion in walking vertebrates. The preparation consists of the first 5 segments of the cervical spinal cord and the attached forelimb. The preparation is bathed in a cooled (15°C) and oxygenated spinal cord Ringers solution and remains viable for 36–100 h. Locomotion can be elicited during the first 36–48 h by applying the excitatory amino acid N-methyl) d-aspartate (NMDA) to the bath. Cutaneous and dorsal root reflexes remain unchanged for much longer periods of time (72–100 h). During locomotion, intracellular recordings can be made from interneurons and motoneurons while simultaneous electromyographic (EMG) recordings are made from forelimb muscles. Rhythmically active interneurons can be classified according to their phase of activity during the step cycle. Further classification of interneurons involves both monitoring the afferent input to these cells from dorsal root and cutaneous afferents as well as using their action potentials as a trigger for averaging the ongoing locomotor EMG activity. In this way some of the input and output characteristics of the interneurons can be monitored. The ability to record simultaneously from interneurons and muscles offers distinct advantages over current in vitro preparations.