Safe and efficient use of spinal drugs requires neurotoxicologic animal studies before ethical application. We have evaluated the neurotoxicologic interruptions of intrathecal administration of midazolam in rabbits. Eighteen white New Zealand rabbits were randomly assigned into three groups consisting of six rabbits each. In conscious animals, 0.3 ml 0.9% normal saline solution, 0.3 ml 0.1% midazolam (Roche, Dormicum) or 0.3 ml preservative free midazolam were intrathecally administered. Light and fluorescence microscopy evaluations were performed on transverse spinal cord sections by a neurohistopathologist in a blind fashion. Midazolam and preservative free midazolam treated rabbits showed significant histologic changes in light and fluorescence microscopy. The histologic and vascular lesions with the use of midazolam and preservative free midazolam suggested neurotoxic effects; thus chronic intrathecal administration of midazolam should be avoided in humans.