Axonal guidance mechanisms involved in pathway formation by avian spinal interneurons have been studied using a variety of different approaches. The earliest developing spinal interneurons can be divided into two groups on the basis of the initial direction of axonal outgrowth: circumferential cells (C-cells) and primitive longitudinal cells. Perturbation studies suggest that a chemotropic-like mechanism plays a role in the guidance of the C-cell axons toward the floor plate. The shape of floor plate cells is polarized mediolaterally (direction of axon growth) and only in this region do growth cones express integrin and contact the basement membrane. Pathway formation following dorsal to ventral spinal cord rotation indicates that the ventrolateral region of the neural tube may provide a permissive substrate for longitudinally advancing growth cones. In addition, following rostrocaudal rotation of three segments of the neural tube, the axons of dorsolateral border cells in the rotated segments modified their directionality according to the general rostrocaudal polarity of the embryo. Changes in cell adhesion molecule expression by the C-cell growth cones and axons is region and age specific. These observations suggest that a variety of mechanisms and cues are working in the guidance of spinal interneurons in each region of the pathway.