Recent results have demonstrated complete anatomical and functional repair of descending brainstem-spinal projections in chicken embryos that underwent thoracic spinal cord transection prior to embryonic day 13 (E13) of the 21 d developmental period. To determine to what extent axonal regeneration was contributing to this repair process, we conducted experiments using a double retrograde tract-tracing protocol. On E8-E13, the upper lumbar spinal cord was injected with the first fluorescent tracing dye to label those brainstem-spinal neurons projecting to the lumbar cord at that time. One to two days later (on E10-E15), the upper to mid-thoracic spinal cord was completely transected. After an additional 7-8 d, a different second fluorescent tracing dye was injected into the lumbar cord at least 5 mm caudal to the site of transection. Finally, 2 d later on E19 to postnatal day 4, the CNS was fixed and sectioned. Brainstem and spinal cord tissue sections were then viewed with epifluorescence microscopy. In comparison to nontrasected control animals, our findings indicated that there were relatively normal numbers of double-labeled brainstem-spinal neurons after a transection prior to E13, whereas the number of double- labeled and second-labeled brainstem-spinal neurons decreases after an E13-E15 transection. In addition, at each subsequent stage of development from E10 to E12, there was a greater number of double- labeled brainstem-spinal neurons (indicating regeneration of previously severed axons) than cell bodies labeled with the second fluorescent tracer alone (indicating subsequent development of late brainstem- spinal projections). Assessment of voluntary open-field locomotion (hatchling chicks) and/or brainstem-evoked locomotion (embryonic or hatchling) indicated that functional recovery of animals transected prior to E13 was indistinguishable from that observed in control chicks (sham operated or unoperated). Taken together, these data suggest that regeneration of previously axotomized fibers contributes to the observed anatomical and functional recovery after an embryonic spinal cord transection.