The in vitro whole spinal cord preparation has been an invaluable tool for the study of the neural network that underlies walking, as it provides a means of recording fictive locomotor activity following surgical and/or pharmacological manipulation. The recent use of molecular genetic techniques to identify discrete neuronal populations in the spinal cord, and subsequent studies showing some of these populations to be involved in locomotor activity, have been exciting developments that may lead to a better understanding of the structure and mechanism of function of this neural network. It would be of great benefit if the in vitro whole spinal cord preparation could be updated to allow for the direct targeting of genetically-defined neuronal populations, allowing each to be characterized physiologically and anatomically. This report describes a new technique that enables the visualization of, and targeted whole cell patch clamp recordings from, genetically-defined populations of neurons while leaving connectivity largely intact. The key feature of this technique is a small notch cut in the lumbar spinal cord which reveals cells located in the intermediate laminae while leaving the ventral portion of the spinal cord, the region containing the locomotor neural network, untouched. Whole cell patch clamp recordings demonstrate that these neurons are healthy and display large rhythmic depolarizations that are related to electroneurogram bursts recorded from ventral roots during fictive locomotion. Intracellular labeling demonstrates that this technique can also be used to map axonal projection patterns of neurons. We expect that this procedure will greatly facilitate electrophysiological and anatomical study of important neuronal populations that comprise neural networks throughout the central nervous system.