Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. Here, we show that DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.