Activation of epidermal growth factor receptor (EGFR)-mediated signaling has been implicated in the pathogenesis of tobacco smoke-induced cancers. Recently, elevated levels of amphiregulin, a ligand of the EGFR, were found in the oral mucosa of smokers. The main objective of this study was to elucidate the mechanism by which tobacco smoke induces amphiregulin. Treatment of a nontumorigenic human oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke stimulated amphiregulin (AR) transcription resulting in increased amounts of amphiregulin mRNA and protein. Tobacco smoke stimulated the cyclic AMP (cAMP)-->protein kinase A (PKA) pathway leading to increased cAMP-responsive element binding protein-dependent activation of AR transcription. These inductive effects of tobacco smoke were dependent on the aryl hydrocarbon receptor (AhR). In fact, alpha-naphthoflavone, an AhR antagonist, blocked tobacco smoke-mediated induction of binding of cAMP-responsive element binding protein to the AR promoter and thereby suppressed the induction of amphiregulin. Notably, treatment of MSK-Leuk1 cells with tobacco smoke or exogenous amphiregulin stimulated DNA synthesis. An inhibitor of EGFR tyrosine kinase or a neutralizing antibody to amphiregulin abrogated the increase in DNA synthesis mediated by tobacco smoke. Taken together, these findings suggest that tobacco smoke stimulated a signaling pathway comprised of AhR-->cAMP-->PKA resulting in enhanced AR transcription and increased DNA synthesis. The ability of tobacco smoke to induce amphiregulin and thereby enhance DNA synthesis is likely to contribute to the procarcinogenic effects of tobacco smoke.