Abstract There is increasing evidence for steroid hormone effects in lung cancer. Estrogens are involved in lung cancer proliferation and progression, and human lung tumors express estrogen receptor β (ERβ) as well as aromatase, the enzyme which catalyzes the final step in 17-β-estradiol synthesis. We have previously demonstrated that high ERβ expression in lung tumors was an independent negative prognostic predictor of survival, and the contribution of aromatase expression resulted in further effects on survival in both men and women. To determine if the aromatase inhibitor anastrozole prevents the development of lung tumors induced by the tobacco carcinogen, NNK, alone or in combination with the ER antagonist fulvestrant, ovariectomized FVB/N female mice were exposed to NNK along with daily supplementation of 0.1mg androstenedione, the substrate for aromatase. Placebo control, anastrozole (0.1mg/kg/day; oral gavage) and/or fulvestrant (30mg/kg, 2X/week, s.c.) were administered in both an initiation model and a progression model of lung tumorigenesis. In the initiation model, the combination of fulvestrant and anastrozole was administered during NNK exposure and resulted in significantly fewer NNK-induced lung tumors (mean=0.5; range 0-1) compared to placebo (mean=4.6; range 2–8, P<0.001), fulvestrant alone (mean=3.4; range 1–4, P<0.001) or anastrozole alone (mean=2.8; range 0–5, P=0.002). Although a significant decrease in tumor size was not observed among treatment groups, a significant decrease in the cell proliferation index as assessed by Ki67 immunostaining within the tumors treated with anti-estrogens was observed. Beginning anti-estrogen treatment 9 weeks after NNK exposure in the lung tumor progression model, when preneoplastic areas had already formed, also yielded maximum anti-tumor effects with the combination group. In this model, tumor size was modulated by hormonal treatment with the smallest tumors observed in the combination treated group. These results suggest that targeting the estrogen pathway can inhibit lung cancer development during induction of damage to the airways by tobacco carcinogen exposure as well as after damage has occurred. In both animal models, aromatase expression was found mainly in macrophages infiltrating preneoplastic and tumor cells of the lungs, while ERβ was found in both macrophages and tumor cells. An important source of estrogen synthesis may be inflammatory cells that infiltrate the lungs in response to carcinogens, beginning early in the carcinogenesis process. ERβ expressed by inflammatory and neoplastic epithelial cells in the lung may signal in response to local estrogen production. In summary, the effects of combining two endocrine agents with different modes of actions suggest that complete blockade of estrogen action by inhibition of estrogen synthesis and down-regulation of ER has a greater effect on the prevention of tobacco carcinogen-induced murine lung tumorigenesis, and may be useful for lung cancer prevention in both males and females. Additionally, a greater understanding of the inflammatory state of the lung in relation to estrogen signaling is necessary to understand the early stages of lung tumorigenesis. This work was supported by NIH P50CA090440, SPORE in Lung Cancer to JMS and in part by the Comprehensive Cancer Center award P30CA047904.