The activity of motile protrusions of the growth conefilopodia, veils, and lamellipodia-is essential for directed growth of a neuronal process. The regulation of the formation of these protrusions is not well understood. Numerous filopodia and veils or lamellipodia form within minutes of transection of an Aplysia axon in culture, as the initial components of growth cones of regenerating neurites. Axotomy, therefore, provides a robust and reliable protocol for analyzing the formation of these protrusions. We evaluated the involvement of protein phosphorylation in the regulation of protrusive activity. Of the inhibitors of protein kinases assayed, only the inhibitors of protein tyrosine kinases-genistein, lavendustin A, herbimycin A, and erbstatin analogue-suppressed the formation of protrusions, as assessed by high magnification video microscopy. These drugs did not work by preventing resealing of the axon, as evident from visual inspection and by the unimpaired effectiveness of genistein or lavendustin in preventing formation of filopodia when applied after resealing. Inhibition of protein tyrosine kinases not only prevented the formation of actinbased protrusions, but also caused deterioration of the actin network underlying the protrusive area of preexisting growth cones. Consistent with an involvement of protein tyrosine phosphorylation in the generation of protrusive structures, immunocytochemistry revealed that aggregates of phosphotyrosine appeared at the margins of the axon, from which protrusions emerge shortly after axotomy. These results suggest a role for protein tyrosine phosphorylation in the formation and maintenance of actin-baSed prOhSiVe StrUCtUreS.