Recent studies have shown that estrogens promote the growth of lung cancer cells and may potentially be responsible for increased susceptibility to lung cancer in women. These observations raise the possibility of using antiestrogens in treating and preventing lung cancer. However, it is not clear how estrogen receptors (ERs) modulate the growth of non-small cell lung cancer (NSCLC) cells. Our Western blotting and real-time PCR analysis showed that NSCLC cells expressed ERbeta, but not ERalpha. In addition, ERbeta-specific ligands, but not ERalpha-specific ligands, promoted the growth of lung cancer cells. Furthermore, knockdown of ERbeta by short hairpin RNA constructs resulted in loss of estrogen-dependent growth of lung cancer cells. Interestingly, endogenous ERbeta failed to transcriptionally activate estrogen response element (ERE)-luciferase constructs in NSCLC cells, suggesting a lack of genomic function. Upon further investigation, ERbeta was found to be in the cytoplasm in all lung cancer cells and failed to translocate to the nucleus in the presence of estrogen, as observed by biochemical, ArrayScan, and confocal microscopy experiments. Nonetheless, estrogen caused rapid activation of cAMP, Akt, and MAPK signaling pathways in lung cancer cells. Immunohistochemical analysis of lung tumor biopsies showed strong ERbeta staining in the cytoplasm, whereas no staining was observed for ERalpha. In conclusion, our results suggest that that proliferative effects of estrogen in lung cancer cells is mediated primarily, if not exclusively, by the nongenomic action of ERbeta.