Fetal spinal cord transplants prevent the retrograde cell death of immature axotomized central nervous system (CNS) neurons and provide a terrain which supports axonal elongation in the injured immature spinal cord. The current experiments were designed to determine whether the axons which grow across the site of the neonatal lesion and transplant are derived from axotomized neurons and are therefore regenerating or whether the axons which grow across the transplant are late-growing axons that have not been axotomized directly. We have used an experimental paradigm of midthoracic spinal cord lesion plus transplant at birth and temporally spaced retrograde tracing with the fluorescent tracers fast blue (FB) and diamidino yellow (DY) to address this issue. Fast blue was placed into the site of a spinal cord hemisection in rat pups less than 48 h old. After 3-6 h to allow uptake and transport of the tracer, the source of fast blue was removed by aspiration and the lesion was enlarged to an "over-hemisection." A transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. The animals survived 3-6 weeks prior to the injection of the second tracer (DY) bilaterally into the host spinal cord caudal to the lesion plus transplant. Neurons with late-developing axons would not be exposed to the first dye (FB), but could only be exposed to the second tracer, diamidino yellow. Thus, neurons with a diamidino yellow-labeled nucleus are interpreted as "late-developing" neurons. Neurons axotomized by midthoracic spinal cord lesion at birth could be exposed to the first tracer, fast blue. If after axotomy they regrew caudal to the transplant, they could be labeled by the second tracer as well. We interpret these double-labeled neurons as regenerating neurons. If neurons labeled with fast blue and axotomized by the spinal cord hemisection either failed to regenerate or grew into the transplant but not caudal to it, they would be labeled only by the first dye. We have examined the pattern and distribution of single (FB or DY)- and double (FB + DY)-labeled neurons in the sensorimotor cortex, red nucleus, locus coeruleus, and raphe nuclei. The sensorimotor cortex contains only DY-labeled neurons. The red nucleus contains both FB- and FB + DY-labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)