We investigated the interaction of MAP-2c and Fyn in the initiation of process outgrowth in COS7 cells. Single transfections of Fyn and MAP-2c resulted in a dramatic decrease in flat, rounded COS7 cells, and a significant increase in both the number of cells with multiple short, spike-like processes, and cells with longer processes. Co-transfection of Fyn and MAP-2c resulted in an additive increase in the number of cells with more than two processes and discrete sites of co-localization within processes. When single or double transfected cells were treated with cytochalasin D or lantrunculin there was a dramatic increase in the number of cells with more than two processes. In addition, there was an increase in the length of the processes, both in single and double transfected cells, suggesting that the actin meshwork provides a barrier for MT-based process extension. When co-transfected cells were post-treated with nocodazole, Fyn was not associated with MAP-2c and acetylated, stable tubulin. Although some Fyn/MAP-2c co-localization was retained, punctate staining of MAP-2c and Fyn were observed at the cell periphery, in areas devoid of stable MTs. Mutations in either tyrosine 67 (Tyr67), a site on human MAP-2c phosphorylated by Fyn, or a second tyrosine residue (Tyr50), did not alter the ability of MAP-2c and Fyn to induce process outgrowth. These studies suggest that independent of one another MAP-2c and Fyn are able to induce process outgrowth and in concert can initiate and enhance process outgrowth in an additive manner.