Doxorubicin, a fluorescent cytotoxic antibiotic, was found to be both a retrograde neuron pathway tracer and neurotoxin to cells retrogradely labeled with it. Doxorubicin was injected into rat caudate-putamen and within 4 h the nuclei in the ipsilateral substantia nigra zona compacta (SNc) and ventral tegmental area (VTA) were stained with red fluorescent doxorubicin. After 2 weeks, portions, but not all of the ipsilateral SNc and VTA were depleted of neurons. Retrograde neurotoxicity was obvious following injections of 20%, 10%, 6%, 5% or 4% doxorubicin but not after 1% or 2%. Five months following doxorubicin treatment, the ipsilateral SNc and VTA were shrunken, distorted and nearly absent; the injected caudate was shrunken and replaced by ventricle. The ipsilateral thalamic parafasicular center median nucleus, a complex nucleus also known to project to the caudate, was depleted of large neurons 2 weeks following caudate injection. Doxorubicin can be transported over relatively long distances; lumbar spinal cord injections labeled cortical pyramidal neurons 3 days later. Doxorubicin's unique pathway-specific neurotoxicity may be useful in future neuroscientific studies.