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During wound healing, keratinocytes initiate migration from the wound edge by extending lamellipodia into a fibronectin-rich provisional matrix. While lamellipodia-like structures are also found in cultured keratinocytes exposed to epidermal growth factor (EGF), the signaling pathway that regulates the formation of these structures is not defined. In cultured human keratinocytes seeded on fibronectin, we found that protein-serine/threonine kinase inhibitors including staurosporine, induced concentration-dependent formation of extended lamellipodia (E-lams). The formation of E-lams was inhibited by the proteintyrosine kinase inhibitors herbimycin A and genistein and augmented by the protein-tyrosine phosphatase inhibitor sodium orthovanadate. Staurosporine treatment induced relocation of tyrosine phosphorylated phospholipase C-gamma1 (PLC-gamma1) to the tips of lamellipodia where actin assembly was initiated. Consistent with an involvement of PLC-gamma1 in E-lam formation, intracellular free calcium (Ca2+) was elevated during the formation of E-lams and conversely, E-lam formation was blocked by intracellular Ca2+ chelation with BAPTA/AM, but not by extracellular reduction of Ca2+ by EGTA. Notably, glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) was activated by staurosporine as evidenced by reduced phosphorylation on Ser-21/9. Suppression of GSK-3 activity by LiCl2 or by a specific chemical inhibitor, SB-415286, blocked E-lam formation but without altering cell spreading. Furthermore, GSK-3 inhibitors blocked both staurosporine- and EGF-induced keratinocyte migration in scratch-wounded cultures. We propose that GSK-3 plays a crucial role in the formation of long lamellipodia in human keratinocytes and is potentially a central regulatory molecule in epithelial cell migration during wound healing.