Activation of the epidermal growth factor receptor (EGFR) is a key signaling event that promotes cells to move and cover wounds in many epithelia. We have previously shown that wounding activates the EGFR through activation of the Src family kinases (SFKs), which induce proteolytic shedding of epidermal growth factor-like ligands from the cell surface. A major goal in wound healing research is to identify early signals that promote motility, and here we examined the hypothesis that members of the focal adhesion kinase family are upstream activators of the SFKs after wounding. We found that focal adhesion kinase is not activated by wounding but that a different family member, Pyk2 (PTK2B/RAFTK/CAKbeta), is activated rapidly and potently. Pyk2 interaction with c-Src is increased after wounding, as determined by co-immunoprecipitation experiments. Disruption of Pyk2 signaling either by small interfering RNA or by expression of a dominant negative mutant led to inhibition of wound-induced activation of the SFKs and the EGFR, and conversely, overexpression of wild-type Pyk2 stimulated SFK and EGFR kinase activities in cells. In wound healing studies, Pyk2 small interfering RNA or dominant negative inhibited cell migration. These results show that activation of Pyk2 is an early signal that promotes wound healing by stimulating the SFK/EGFR signaling pathway.