We have investigated gene transfer to the injured adult rat spinal cord by the use of a recombinant adenovirus. 105 or 5 × 106 plaque-forming units (pfu) of a replication-defective adenoviral vector carrying the green fluorescent protein (GFP) reporter gene were injected into a dorsal hemisection lesion at spinal level T8. Gene expression and inflammatory responses were studied 4, 8 and 21 days after surgery. Numerous cells within 3 mm on each side of the lesion were found to express high levels of GFP at 4 days after infection as shown by GFP fluorescence and immunohistochemistry. At 8 days, expression was still strong although weaker than at 4 days. After 21 days, transgene expression had almost ceased. Expression was neither higher nor more prolonged in animals that had received the higher vector dose. Delayed injection 1 week after spinal injury also did not increase transgene expression. Infected cell types were identified immunohistochemically. The most prominent transduced cells were spinal motoneurons. Additionally, we could identify other neurons, astrocytes, oligodendrocytes and peripheral cells infiltrating the lesion site. The glial and inflammatory reaction at and around the lesion was studied by cresyl violet histology, α-GFAP, OX42 and α-CD-8 immunohistochemistry. No significant differences from controls were found in the low virus group; in the high virus group a strong invasion of CD-8-positive lymphocytes was found. Open-field locomotion analysis showed virus-infected animals performing as well as control animals. Adenoviral gene transfer may be an efficient way to introduce factors to the injured spinal cord in paradigms of research or therapy.