Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs the in situ path of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.