Direct routes of gene administration (intrathecal, intracerebroventricular or intraparenchymal infusion) have been used for effective and sustained gene delivery, but serious concerns exist about possible traumatic injury as well as neural damage that may lead to further tissue necrosis, apoptosis and cell death. We evaluated targeted retrograde gene delivery through the sternomastoid muscle (innervated by the spinal accessory nerves) into the injured cervical spinal cord using a recombinant adenovirus vector. LacZ gene expression in the cervical spinal cord was noted from 3 days to 4 weeks after the injection of vector into the sternomastoid muscles of the rats. Recombinant adenovirus vector was transferred via a retrograde mechanism into the injured cervical spinal cord with high transduction efficacy (80.6--98.9%) over certain adenoviral titer and dosage. Transduction was less efficient when the vector was injected 1 and 2 weeks after spinal cord injury (44.2--56.8%). Our results indicate retrograde delivery of recombinant adenovirus vector is possible immediately after spinal cord injury, and that this method is promising for gene delivery because it is effective, selective, less invasive to the injured spinal cord, has long-lasting gene expression, and is potentially feasible treatment choice for spinal cord injury.