Aim: To test the hypothesis that the epoxyeicosatrienoic acid (EET)-induced transactivation of EGF-R depends on the activation of metalloproteinases and the subsequent release of HB-EGF in cancer cells. Methods: Exogenous 14,15-EET were added to four human-derived cancer cell lines Tca-8113, A549, HepG2, and MDA-MB-231, or these same cell lines were transfected with a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase). The effects of elevated EET levels on the phosphorylation of tyrosine residues in the EGF receptor and on ERK1/2 activation were then assessed. Results: Both the addition of 14,15-EET and the transfection of cells with CYP102 F87V markedly increased the phosphorylation of the tyrosine residues of EGF-R and ERK1/2, an effect that was blocked by a selective EGF-R tyrosine kinase inhibitor (tyrphostin AG1478), a broad-spectrum metalloproteinase inhibitor (1,10-phenanthroline), and an inhibitor of HB-EGF release (CRM197) in Tca-8113 cells. In addition, AG1478, 1,10-phenanthroline, and CRM197 also inhibited the tyrosine phosphorylation of EGF-R and ERK1/2 that was induced by 14,15-EET in the A549, HepG2, and MDA-MB-231 cell lines. Conclusion: These results suggest that the EET-induced transactivation of EGF-R depends on activation of metalloproteinases and the subsequent release of HB-EGF in cancer cell lines.