An in vitro preparation was described for studying electrical activity of mammalian brain stem and spinal cord. The brain stem and the spinal cord were isolated from 0-4-day-old rats, placed in a bath and perfused with modified Krebs solution. Various reflex responses could be recorded from cranial nerves by stimulation of other cranial nerves. The preparation was viable for more than 7 h. Spontaneous periodic neural activity could be recorded from phrenic, hypoglossal and other spinal nerves. The periodic discharges of phrenic nerves are synchronized with those of ventral roots C4 and the upward movements of the thorax which was isolated together with the spinal cord. The rhythm of periodic activity seems to be generated in the brain stem. The periodic activity was enhanced by perfusion with low pH solution and depressed by high pH solution. It was markedly depressed by opioid compounds such as enkephalin. It is suggested that this periodic activity corresponds to the respiratory rhythm generated in the brain stem of intact animals. The present preparation may be valuable for elucidating cellular mechanisms of generation and control of respiratory rhythm in the mammalian central nervous system.