Previously, we identified the serine/threonine kinase Akt as a molecular target in lung cancer, because Akt is constitutively active in non-small cell lung cancer cells in vitro and in vivo, and tobacco components activate Akt in primary cultures of human lung epithelial cells. To determine a role for Akt activation in the transformation of lung epithelial cells, we evaluated the PI3K/Akt pathway in immortalized human lung epithelial cells (BEAS-2B cells) or in BEAS-2B cells that were fully transformed by a tobacco-specific carcinogen (ie, BEAS-2B/4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone [NNK] cells) in vitro, as well as NNK-induced murine lung lesions in vivo. Adding nicotine or NNK to BEAS-2B or BEAS-2B/NNK cells increased Akt activation in a time-dependent and dose-dependent manner, and increased cellular proliferation. Compared to immortalized BEAS-2B cells, tumorigenic BEAS-2B/NNK cells had higher levels of epidermal growth factor receptor (EGFR) phosphorylation, increased Akt activity, increased phosphorylation of multiple substrates downstream of Akt, and enhanced survival under conditions of serum starvation. Pharmacologic inhibitors of PI3K, Akt, or the EGFR decreased Akt activity and increased apoptosis of BEAS-2B and BEAS-2B/NNK cells, with the greatest relative induction of apoptosis observed with BEAS-2B/NNK cells.