We have shown that the tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an agonist for-adrenergic receptors (beta-ARs) and increased DNA synthesis of human lung adenocarcinoma cells with features of bronchiolar Clara cells by binding to these receptors. Using a cell line derived from a human pulmonary adenocarcinoma with Clara cell phenotype (PACC) and immortalized human small airway epithelial cells (HPLD1), the putative cells of origin of this cancer type, our current studies have analyzed signaling initiated by binding of NNK to the beta 1-AR. NNK upregulated ERK1/2 and CREB/ATF-1 phosphorylation in a PKA-dependent manner in both cell lines. This response was further increased by transient overexpression of the beta 1-AR. Pre-exposure of cells to the selective beta 1-AR antagonist, atenolol, attenuated the stimulatory effects of NNK, suggesting the latter upregulated ERK1/2 and CREB/ATF-1 via this receptor. In vivo labeling and immunoprecipitation assays revealed that NNK phosphorylated the epidermal growth factor receptor (EGFR) at tyrosine residues, 991, 1068 and 1173, an effect inhibited by atenolol. The inhibitor of EGFR-specific tyrosine kinases, AG1478, reduced NNK ability to stimulate ERK1/2 and CREB/ATF-1. Genomic analysis of the exons 18-21 of the EGFR genes showed that no mutations were present in either gene. Collectively, our data provide evidence, for the first time, that NNK targets ERK1/2 and CREB/ATF-1 proteins via dual signaling involving beta 1-AR and EGFR pathways in PACCs and their putative cells of origin.