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Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical (C5-T2) and lumbar (L3-6) segments of adult guinea pigs. The spinal cord was cut into serial frozen sections. The Fluoro-ruby labeling was clearly delineated from the surrounding structure. The labeling traversed the cervical, thoracic and lumbar segments, and was located on the ventral portion of the posterior funiculus on the injected side, proximal to the intermediate zone of the dorsal gray matter. The fluorescence area narrowed rostro-caudally. The spinal cord, spinal cord gray matter and corticospinal tract were reconstructed using 3D-DOCTOR 4.0 software, resulting in a robust three-dimensional profile. Using functionality provided by the reconstruction software, free multi-angle observation and sectioning could be conducted on the spinal cord and corticospinal tract. Our experimental findings indicate that the Fluoro-ruby retrograde fluorescent tracing technique can accurately display the anatomical location of corticospinal tract in the guinea pig and that three-dimensional reconstruction software can be used to provide a three-dimensional image of the corticospinal tract.