We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn(2+)-induced EGFR activation. Exposure to Zn(2+) induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn(2+) on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn(2+)-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn(2+)-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn(2+)-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn(2+)-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn(2+) induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.