Localized expression of environmental cues is thought to provide directional information to migrating neuronal growth cones by enhancing or suppressing axon outgrowth over limited regions. To investigate how such a mechanism may function in vivo, we observed growth cones of embryonic chick dorsal root ganglion neurons at a substratum border between the extracellular matrix components laminin and fibronectin in vitro using time-lapse phase-contrast and interference reflection microscopy. We found that patterns of laminin and fibronectin could locally promote or suppress the direction of growth cone migration. While migrating on either laminin or fibronectin, at least 79% of growth cones changed their rate and/or direction of outgrowth upon contact with the alternative substratum, in a manner suggesting that growth cones were selecting one substratum over the other. Complex changes in growth cone behavior were initiated by filopodial contact with the alternate substratum, suggesting that filopodia were providing intracellular signals to the growth cone. Using interference reflection microscopy, we have found that selection of a substratum is independent of the degree of close contact to the substratum. We conclude that spatially localized ECM components can direct axon outgrowth by mechanisms based on intracellular signaling through growth cone filopodia.