Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. Here we report that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. We detected that stimulation of DCC receptors with netrin-1 resulted in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development.Netrins are a small family of secreted proteins that guide growing axons during neural development (1, 2). The first netrin cloned, UNC-6, was identified using a genetic screen for mutations affecting axon guidance in Caenorhabditis elegans (3). Netrins were first identified in vertebrates on the basis of their ability to promote commissural axon outgrowth from explants of embryonic spinal cord (4, 5). Netrin family members have now been identified in multiple vertebrate and invertebrate species and shown to have a highly conserved function as axon guidance cues (6). Netrins are bifunctional molecules attracting and repelling different classes of axons. Growth cone attraction mediated by netrin-1 involves the transmembrane netrin receptor DCC (7,8). In C. elegans, the identification of UNC-5 first implicated it as a receptor required for the repellent response to UNC-6 (9). Three UNC-5 homologs have now been identified in mammals (10, 11). Current evidence suggests that netrin-mediated repulsion requires the function of both UNC-5 and DCC family members in some and perhaps all cases, suggesting that UNC-5 and DCC may form a netrin receptor complex (12).The intracellular mechanisms mediating the response of an axon to netrin-1 are currently unclear. Previous studies indicate that extracellular guidance cues induce the neuronal growth cone to advance, retract, or turn by regulating the actin cytoskeleton within the growth cone (13). The Rho family of small GTPases, in particular, RhoA, Rac1, and Cdc42, are well established regulators of actin reorganization in non-neuronal cells (14), and there is now compelling evidence demonstrating roles for RhoA, Rac1, and Cdc42 as signaling elements within the neuronal growth cone (15, 16). Here, we report that members of the Rho family of GTPases are required for commissural axon outgrowth produced by netrin-1 from explants of embryonic rat spinal cord. Both Rac1 and Cdc42 are required for neurite outgrowth promoted by DCC ...