Application of neurotrophic factors (NFs) to the cut stump of peripheral nerves confers transient (1- to 2-week) neuroprotection of motoneurons from axotomy-induced death in neonates. We tested whether lumbar spinal motoneurons would be protected from axotomy-induced death when they were genetically modified to produce NFsin situ. Adenoviral (Adv) vectors carrying neurotrophic factor genes under control of the Rous sarcoma virus long terminal repeat promoter (Adv.RSV-nf) or a control vector containing the β-galactosidase (β-gal) gene (Adv.RSV-βgal) was injected into the hindlimb muscles of neonatal rats. The Adv were taken up by peripheral nerves and transported to lumbar spinal cord motoneurons where the transgenes were expressed. A fraction (18%) of the motoneurons that projected through the sciatic nerve were transduced with Adv.RSV-βgal. Expression of Adv.RSV-βgal was detected in motoneurons after 7 days and 3 weeks, with no evidence of vector- or β-gal-induced toxicity or inflammation. PCR, immunocytochemistry, and RT-PCR demonstrated transport of the Adv.RSV-nfvectors to motoneurons and their expression. After retrograde transport of an Adv.RSV-nfvector carrying the gene for glial cell line-derived neurotrophic factor, a substantial proportion of the sciatic nerve motoneurons were resistant to axotomy-induced death 7 days and 3 weeks after sciatic nerve transection (56 and 44%, respectively), compared to Adv.RSV-βgal controls (2.5 and 0%, respectively).