Mammalian spinal cord motoneurons are highly susceptible to chemical and mechanical disturbances, which imposes substantial difficulties for electrophysiological investigation in acute in vitro preparations. The aim of the present study was to establish an isolated spinal cord preparation from adult mice and to examine the synaptic activities of motoneurons in vitro. We removed the lumbo-sacral cord from the vertebral canal by hydraulic extrusion and maintained the isolated cord in vitro for extracellular recordings. Population spikes of motoneurons were evoked by electrical stimulation of dorsal roots (orthodromic) or ventral roots (antidromic) and these evoked responses could be continuously monitored for 5-6 h. The orthodromic population spikes were reversibly suppressed by the AMPA/kainate receptor antagonist 2,3-dihyro-6-nitro-7-sulfamoylbenzo quinoxaline (NBQX, 10 microM) but they persisted in the presence of the NMDA receptor antagonist D(-)-2-amino-5-phosphonovaleric acid (AP5, 50 microM). The antidromic population spikes exhibited evident paired pulse inhibition when evoked at inter-stimulus intervals of pound 6 ms. Histological examination revealed that basic morphological features of the lumbo-sacral motoneurons were preserved after 3-4 h of in vitro maintenance. This in vitro preparation is ideally suited for the electrophysiological study of synaptic transmission on adult mouse spinal motoneurons.