To clarify axonal guidance mechanisms involved in pathway formation by spinal interneurons, the pattern of axonal outgrowth was examined following three kinds of perturbations: (1) rotation of three segments of the neural tube around the dorsal-ventral axis by 90 degrees or 180 degrees (D-V rotation), (2) rotation of three segments of the neural tube around the rostral-caudal axis (R-C rotation), and (3) transplantation of brainstem into thoracic spinal segments (BS transplantation). Following D-V rotation, it was observed that circumferential axons near the junction between rotated graft (RG) and host cord changed their course so as to project toward the ectopic floor plate. This supports the notion that the floor plate exerts a chemotropic-like effect on the directional projection of circumferential axons. The longitudinal fibers in the ventral funiculus (VF) were able to grow through the transplant junction when the VF was well apposed to the VF of the RG. However, in most cases, the longitudinal fibers in the VF that were not apposed to the VF of the RG made a medial turn at the junction. After turning, some of them made a U-turn into the contralateral VF, whereas others grew circumferentially around the junction zone, then exited the spinal cord; still others directly entered the RG. The majority of fibers that entered the RG were located in the VF, though a small number of such fibers were also located in both the lateral and dorsal funiculus (LF and DF, respectively). The fibers that entered the LF shifted ventrally, whereas the fibers in the DF remained within the DF. These results suggest that there may be some matching mechanism between specific fibers and the funiculus through which the fibers normally project. Moreover, there may be a barrier between the LF and the DF that prevents the fibers in the DF from shifting ventrally. In R-C rotation experiments, the projection of axons of dorsolateral border cells (DLB- cells), which are known to give rise almost exclusively to commissural ascending axons, was examined. Following the injection of HRP into the rostral junctional region, retrogradely labeled DLB-cells were observed in the rotated segments only on the side contralateral to the injection. Labeled DLB-cells were also observed in the segments caudal to the rotated segments. These results suggest that putative rostrocaudal directional cues that guide the axons of DLB-cells may arise from outside of the segments in which the decision to turn is made.