This study was aimed at developing a method of organotypic culture of spinal cord slice. The slice culture was prepared using lumbar spinal cord from 8-day-old rat. The survival of alpha-motor neuron was evaluated by the morphological observation and by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker and immunohistochemical staining. Lactate dehydrogenase (LDH) level in culture medium was also measured. The result showed that the spinal cord explant could be maintained in the culture for more than 2 months with excellent cellular organization and stable population of ventral motor neurons. The level of LDH at different culture times had no significant difference. Organotypic spinal cord slice culture may provide an effective method for studying physiological and pathological changes, and neuroprotection of spinal cord.