Growth and guidance of developing or regenerating axons require sensing of environmental cues (EC) by the growth cone. To explore the role of a spatially defined distribution of ligands on guidance, extension, and branching, we used a microcontact-printing technique allowing to deposit ligands as discrete spots of a size smaller than a cell body. Micropatterned substrates (MS) were created with varying distance between spots and two different ligands (laminin (LN) and fibronectin (FN)). Dissociated dorsal root ganglion neurons were seeded on either monocomponent MS made from LN or FN alone, or multicomponent MS made from alternating lines of LN and FN spots. On monocomponent MS the high-affinity ligand LN not only stimulated neurite extension, but also provided guidance and branching control, associated with marked cytoskeleton remodeling. The latter was assessed by evaluating the increase in rigidity of the distal neurite segment by Atomic Force Microscopy. In contrast, FN alone acts as a low-affinity ligand which dramatically limits neurite outgrowth. Surprisingly, observation of growth cone dynamics on multicomponent MS revealed that FN constitute a transient support for neurite progression, facilitating exploration of other EC present within a certain distance. Such a mutual contribution of high and low affinity ligands to neurite outgrowth is consistent with a recent theory of force regulation of dynamic adhesion sites showing cell's sensitivity to EC properties would actually depend on the rate of change of the reacting force, the latter controlling the otherwise instantaneous chemical binding process. © 2011 Wiley-Liss, Inc.