In the developing nervous system the modelling of axons and their growth cones is dependent on the dynamic regulation of the Rho family of GTPases, which play a crucial role in the regulation of the actin cytoskeleton. The guidance cues controlling axon path finding, either repulsive or attractive, require the Rho GTPases to effect changes in morphology. The signalling pathways linking the guidance molecules, and their receptors, to the Rho family GTPases remain unclear. Collapsin Response Mediator Protein-2 (CRMP-2) is a neurospecific protein involved in axonal outgrowth and the semaphorin3A collapse pathway. CRMP-2 is also a Rho kinase substrate, suggesting an involvement with the Rho GTPases. To investigate this, CRMP-2 was co-expressed in the neuroblastoma cell line, N1E-115, with active and inactive GTPase mutants. Cells expressing dominant active Rac1 and CRMP-2 became contracted, normally a RhoA effect, while co-expression of dominant active RhoA and CRMP-2 resulted in a phenotype typically associated with Rac1 signalling. CRMP-2 could bind directly to RhoA and Rac1, and, to a much lesser extent, Cdc42 in an overlay assay. In vivo CRMP-2 associated with active RhoA, but immunoprecipitated with active and inactive Rac1 mutants. Cdk5 inhibitors, but not Rho-kinase inhibitors blocked semaphorin3A-induced collapse in dorsal root ganglion neurones and N1E-115. Mutation of the Cdk5 phosphorylation site in CRMP-2 also inhibited sema3A collapse, suggesting a specific role of Cdk5 and CRMP-2 in the semaphorin3A growth cone collapse pathway. These results show CRMP-2 can switch RhoA and Rac1, and may link the guidance cues to the Rho-GTPases, which define growth cone morphology through their regulation of the actin cytoskeleton. Downstream of sema3A, CRMP-2 plays a crucial role in growth cone collapse, in a pathway involving Cdk5, and possibly phosphorylation at serine 522.