The role of estrogens in the increased risk of lung adenocarcinoma in women remains uncertain. We reported that lung adenocarcinoma cell lines from female, but not male, NSCLC patients respond proliferatively and transcriptionally to estradiol (E2), despite equal protein expression of estrogen receptors and (ER and ER). To test the hypothesis that nuclear localization of ER corresponds to genomic E2 activity in lung adenocarcinoma cells from females, cell fractionation, immunoblot, and confocal immunohistochemical microscopy were performed. We report for the first time that E2 increases phospho-serine-118-ER (P-ser118-ER ) and cyclin D1 nuclear colocalization in H1793 but not A549 lung adenocarcinoma cells derived from a female and male patient, respectively. ER was primarily in the cytoplasm and mitochondria, independent of E2 treatment, and showed no difference between H1793 and A549 cells. E2 induced higher transcription of endogenous ER-regulated CCND1 (cyclin D1) in H1793 than in A549 cells. Likewise, higher rapid, non-genomic E2-induced ERK1/2 activation was detected in H1793 compared to A549 cells, linking ERK activation to increased P-ser118-ER. Further, E2 increased cyclin D1 and P-ser118-ER nuclear localization in H1793, but not A549 cells. Together, our results indicate that nuclear localization of P-ser118-ER provides one explanation for gender-dependent differences in E2-genomic responses in lung adenocarcinoma cell lines.