It has been documented that estrogen receptor (ER) transcription silencing due to hypermethylation is linked to the tumor progression of breast, uterine and prostate cancers. Additionally, ER hypermethylation in lung tumors has been associated with the exposure of specific carcinogens in animal study. The role of hypermethylation-induced ER transcription silencing in lung tumor progression and its prognostic value for non-small cell lung cancer (NSCLC) patients remained unclear. In our study, ER hypermethylation of 123 lung tumors and adjacent normal parts were examined by methylation-specific PCR (MSP). Estrogen receptor mRNA expression in lung tumors was determined by RT-PCR. Our data indicated that ER hypermethylation was only detected in lung tumors, but not in adjacent normal lung tissues. This suggests that ER hypermethylation may be associated with lung tumorigenesis. Among the clinical parameters studied, only gender factor was correlated with ER hypermethylation with a higher frequency of ER hypermethylation being in male patients than in female patients (58 vs. 34%, p = 0.01). After being stratified by gender and cigarette smoking status, a similarly high prevalence of ER hypermethylation was found in male smoking and nonsmoking patients (60 vs. 61%) as compared to that of female nonsmoking patients (34%). To investigate if 17-β estradiol (E2) was responsible for such gender difference in ER hypermethylation, a lung cancer A549 cell with ER hypermethylation and without ER mRNA expression was treated with E2 of various concentrations for defined time intervals to show that an E2 treatment could restore the expression of ER mRNA and eliminate ER hypermethylation. Western blot data also showed that acetylated histone 3 and histone 4 of chromatin were increased significantly by E2 treatment. Thus, E2 can make ER mRNA re-expression by eliminating ER hypermethylation. To elucidate the prognostic value of ER hypermethylation, Kaplan-Meier analysis was carried out to show that patients with ER hypermethylation had a poorer prognosis than those without ER hypermethylation. Such prognostic prediction, however, applied only to male (p = 0.0044) patients. Cox regression analysis further showed the feasibility of ER hypermethylation as an independent prognostic factor of NSCLC (p = 0.007). It is possible that antiestrogens may have different therapeutic values for male and female lung cancer patients. © 2005 Wiley-Liss, Inc.