Anesthetic and surgical procedures and an electrophysiological method were developed for recording nerve conduction velocity (NCV) of CNS fibers in the murine spinal cord. Under intravenous anesthesia and artificial ventilation the lumbar spinal cord segments L1 to L4 and dorsal roots L3 to L5 on the left side were exposed by laminectomy. After stimulation of the dorsal root L4, a compound action potential (CAP) was recorded at the ipsilateral left fasciculus gracilis at the spinal cord level L1. The latency from stimulation to the CAP together with the measured distance between the electrodes was used for the determination of the NCV. NCV of the fastest fibers in the fasciculus gracilis was observed to be approximately 28 m/s. Reversible decrease of the NCV was measured, in vivo, under general hypothermia. The technique described serves for in vivo electrophysiological investigations of spinal central fibers in wildtype and mutant mice.