This study was undertaken to establish a method for the culture of organotypic spinal cord slices. A long-term organotypic spinal cord slice culture was conducted from postnatal rats. Lumbar spinal cord was isolated, and meninges were removed from the spinal cord. The spinal cord was embedded in 4% agarose, and was sectioned by vibratome into slices. Then the slices were cultured on the surface of the membrane inserts, which were placed in six-well plates containing 1 ml of growth medium at 37°C in an incubator with 5% humidified carbon dioxide. The cultured organotypic spinal cord slices were examined by light microscopy and immunocytochemistry. The organotypic spinal cord slices were fully attached to the membrane inserts after 10 days in vitro. The general change in color and transparency from whitish to transparent gray appeared at the seventh and eighth day. Under the light microscope, the outgrowth of cells from the edge of the living slices arose from the second day of the culture, and arose to peak at the sixth and seventh day. The organotypic spinal cord slices were characterized as clear, semitransparent structures with bright and good refraction until the 14th day of culture. The viability of the slices was excellent as assessed by the trypan blue exclusion method at the 28th day, and they were positive for NeuN and GFAP. This culture technique, which does not require complex operation skills, might be a simple and efficient method for obtaining organotypic spinal cord slices in sufficient number, high viability, and contamination-free from postnatal rats.