To clarify the afferent innervation of the canine scrotal contents, retrograde labeling of neurons in the dorsal root ganglia (DRG) has been carried out using two methods: (1) horseradish peroxidase (HRP) injection into the surface of the testis and epididymis; and (2) exposure of the superior spermatic nerve to a fluorescent dye (Fast blue; FB). Injections of HRP resulted in labeling of DRG cells located predominantly from T10 to L4 (87%) and, to a lesser extent, at S1-S3 (13%). Transection of the vas deferens previous to testicular injections eliminated labeling in the S1-S3 DRG, but not at thoracolumbar levels. These findings indicated that primary afferent fibers of the testis and epididymis project mainly to the DRG at higher than L4 through the superior spermatic nerve, but an additional population of the fibers also projects the sacral level through the inferior spermatic nerve. Exposure of the superior spermatic nerve to FB resulted in a similar distribution of labeled cells as compared with testicular injections of HRP after vasectomy. Labeled cells (8.1%) were also observed in the contralateral T13-L3 DRG. In both FB and HRP groups, the major part of the labeled cells was located in L1 and L2. The sizes of HRP- and FB-labeled cells were smaller than those of unlabeled cells in the L1 and L2 DRG. The cumulative frequency distribution histogram for the diameter of HRP- and FB-labeled cells could be fitted by a normal distribution.