BACKGROUND: The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth. RESULTS: Expression of alpha4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type alpha4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal alpha4-mediated migration in leucocytes requires spatial regulation of alpha4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this alpha4(S988D), which mimics phosphorylated alpha4, did not promote neurite outgrowth. However, alpha4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable alpha4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. CONCLUSION: We establish the importance of the alpha4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of alpha4 for extension of neuronal growth cones and migration of non-neural cells.