Axon guidance is influenced by the presence of heparan sulfate (HS) proteoglycans (HSPGs) on the surface of axons and growth cones (Hu, : Nat Neurosci 4:695-701 ; Irie et al. : Development 129:61-70 ; Inatani et al. : Science 302:1044-1046 ; Johnson et al. : Curr Biol 14:499-504 ; Steigemann et al. : Curr Biol 14:225-230 ). Multiple HSPGs, including Syndecans, Glypicans and Perlecans, carry the same carbohydrate polymer backbones, raising the question of how these molecules display functional specificity during nervous system development. Here we use the Drosophila central nervous system (CNS) as a model to compare the impact of eliminating Syndecan (Sdc) and/or the Glypican Dally-like (Dlp). We show that Dlp and Sdc share a role in promoting accurate patterns of axon fasciculation in the lateral longitudinal neuropil; however, unlike mutations in sdc, which disrupt the ability of the secreted repellent Slit to prevent inappropriate passage of axons across the midline, mutations in dlp show neither midline defects nor genetic interactions with Slit and its Roundabout (Robo) receptors at the midline. Dlp mutants do show genetic interactions with Slit and Robo in lateral fascicle formation. In addition, simultaneous loss of Dlp and Sdc demonstrates an important role for Dlp in midline repulsion, reminiscent of the functional overlap between Robo receptors. A comparison of HSPG distribution reveals a pattern that leaves midline proximal axons with relatively little Dlp. Finally, the loss of Dlp alters Slit distribution distal but not proximal to the midline, suggesting that distinct yet overlapping pattern of HSPG expression provides a spatial system that regulates axon guidance decisions.