Environmental or occupational exposure to arsenic is associated with a greatly increased risk of skin, urinary bladder, and respiratory tract cancers in arseniasis-endemic areas throughout the world. Arsenic shares many properties of tumor promoters by affecting specific cell signal transduction pathways responsible for cell proliferation. The activation of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated protein kinase (ERK) pathway is important in mediating gene expression related to regulation of cellular growth. In the current studies, we demonstrate that arsenic activates EGFR and ERK in a human uroepithelial cell line. The EGFR phosphorylation by arsenic is ligand-independent and does not involve the major autophosphorylation site Tyr(1173). c-Src activity is also induced by arsenic and is a prerequisite for the EGFR and ERK activation. Consistent with these in vitro observations, exposure of mice to arsenic in drinking water, which has been found previously to be associated with AP-1 activation and epithelial proliferation, induces EGFR and ERK activation in the urinary bladder. This response is also accompanied with an increase in c-Src levels interacting with EGFR. These findings represent a potential pathway for mediating arsenic-induced phenotypic changes in the uroepithelium.