The objective of this study was to investigate the influence of the length of the remaining axons on neuronal survival following axonal injury in the adult rat spinal cord. The relationship between the expression of nitric oxide synthase (NOS) and neuronal death was also studied. The time course of NOS expression and neuronal loss was observed in Clarke's nucleus at segment L1 following hemisection of the cord at either segment C3, T1, or T11. NOS-positive neurons were first observed 3 days after injury, rising significantly after 5 days, and reaching a maximum by 10-20 days post-injury. During the same period the number of neurons surviving in the nucleus decreased. The decrease began at 3 days, rapidly decreased after 5 days, and reached a minimum by 40 days post-injury. Among the three lesioned groups, the highest percentage of NOS expression and neuronal loss was observed in the group with lesions at T11, while the lowest percentage was in the group with lesions at C3. Although the time course of neuronal loss varied following lesion at different spinal levels, in all cases the majority of neurons in Clarke's nucleus had degenerated by 40 days after axotomy.