We previously showed that alpha-synuclein (alpha-Syn), a protein implicated in the pathogenesis of several neurodegenerative diseases, is a microtubule-associated protein (MAP), facilitating the polymerization of tubulin into microtubules. Therefore, we hypothesized that alpha-Syn might promote neurite outgrowth, a process that requires microtubule assembly. To test this hypothesis, recombinant human wild type (WT) and mutant (A30P and A53T) alpha-Syn proteins were added to cultured primary rat cortical neurons, and their effects on early neurite outgrowth were observed. The WT and mutant alpha-Syn proteins entered the neurons after 1-4 h of incubation. However, a significant increase in neurite outgrowth was observed only in neurons treated with WT alpha-Syn. MES23.5 dopaminergic neuronal cells overexpressing WT alpha-Syn also exhibited enhanced neurite outgrowth, indicating that the ability of alpha-Syn to promote neurite outgrowth was not due to a direct action on the cell membrane or by the membrane translocation process. Co-immunoprecipitation demonstrated that the recombinant human alpha-Syn was bound to tubulin. In addition, the alpha-Syn-treated neurons displayed increased levels of polymerized tubulin. Because alpha-Syn's MAP functionality is mediated by specific domains, we generated N-terminal (a.a. 1-65), non-amyloid-beta (non-A beta) component (NAC) (a.a. 61-95) and C-terminal (a.a. 96-140) fragments and added them to the primary neurons. After 1-4 h of incubation, the various alpha-Syn fragments had entered the neurons. However, only the NAC and C-terminal fragments, which have been previously shown to mediate MAP functionality, promoted neurite outgrowth. These results suggest that alpha-Syn promotes neurite outgrowth by facilitating the polymerization of tubulin into microtubules.