Members of the Semaphorin family of glycoproteins play an important role in axonal pathfinding by functioning as inhibitory guidance cues. Here we provide evidence that a transmembrane form of Semaphorin (Semaphorin I), which is expressed by bands of epithelial cells in the developing grasshopper limb bud, functions as an attractive/permissive cue for the growth cones of the subgenual organ. In addition, we demonstrate that Semaphorin I is needed for initial axonal outgrowth from the subgenual organ. These results are consistent with an alternative function for a transmembrane form of Semaphorin and may explain the previously reported arrest of the proximal extension of the subgenual organ growth cones in the absence of the Ti1 pioneer pathway.