In vitro slice preparations of CNS tissue are invaluable for studying neuronal function. However, up to now, slice protocols for adult mammal spinal motoneurons-the final common pathway for motor behaviors-have been available only for limited portions of the spinal cord. In most cases, these preparations have not been productive due to the poor viability of motoneurons in vitro. This report describes and validates a new slice protocol that for the first time provides reliable intracellular recordings from lumbar motoneurons of adult rats. The key features of this protocol are: pre-exposure to 100% oxygen, laminectomy prior to perfusion, anesthesia with ketamine/xylazine, embedding the spinal cord in agar prior to slicing, and, most important, brief incubation of spinal cord slices in a 30% solution of polyethylene glycol to promote re-sealing of the many motoneuron dendrites cut during sectioning. Together, these new features produce successful recordings in 76% of the experiments and an average action potential amplitude of 76 mV. Motoneuron properties measured in this new slice preparation (i.e., voltage and current thresholds for action potential initiation, input resistance, afterhyperpolarization size and duration, and onset and offset firing rates during current ramps) are comparable to those recorded in vivo. Given the mechanical stability and precise control over the extracellular environment afforded by an in vitro preparation, this new protocol can greatly facilitate electrophysiological and pharmacological study of these uniquely important neurons and other delicate neuronal populations in adult mammals.