Arachidonic acid is metabolized to biologically active epoxyeicosatrienoic acids (EETs) by cytochrome P450 (CYP) epoxygenases. Previous studies showed that CYP epoxygenases promote neoplastic growth and induce potent mitogenic effects in human carcinoma cells; however, the exact molecular mechanisms involved in EET-induced tumor cell proliferation remain unclear. Exogenous 14,15-EET was added or a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase) was transfected into three human derived cancer cell lines; Tca-8113, A549, HepG2 and MDA-MB-231. The effects of elevated EETs on tyrosine phosphorylation of the EGF receptor and ERK1/2 activation were then assessed. In this study, we found that addition of 14,15-EET and CYP102 F87V transfection markedly increased tyrosine phosphorylation 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 induced by 14,15-EET in A549, HepG2 and MDA-MB-231 cancer cell lines. These data suggest that EET-induced transactivation of EGF-R depends on activation of metalloproteinases and subsequent release of HB-EGF in cancer cells.