of bisulfite-treated DNA (EpigenDx, Worcester MA). Altogether, we examined the methylation level across 39 CpG sites in the GR exon 1F promoter region for each subject. Two genes were genotyped; the 5HTTLPR and the ESR1. All statistical tests were carried out using SPSS version 15. A linear regression model was used to ascertain the effects of sex, GR methylation level and genes on stress response. Statistical normality of the data was checked using the Kolmogorov-Smirnov test. Results: Marked individual differences were observed in methylation levels of GR exon 1F at individual CpG sites for females and males. Overall, women showed significantly greater methylation levels than did men (t = 2.538, p = 0.013). There was a correlation between total cortisol output (area under the curve, AUC) and average methylation level at GR exon 1F in female subjects (R2D= 0.214, F(1,44) = 11.997, p = 0.001) accounting for 21.4% of the variance. Additionally, variations in the ESR1 and the 5-HTTLPR genes were significant predictors of AUC. A significant main effect of 5-HTTLPR (R2D= 0.172, F1,42 = 12.032, p = 0.001) and ESR1 (R2D= 0.132, F2,40 = 5.634, p = 0.007) was observed on AUC but there was no interaction between methylation and either gene. The full model accounted for nearly half of the variance (48%) in total cortisol output. Conclusions: We provide the first evidence that accumulated epigenetic changes at the peripheral GR exon 1F correlate with HPAA reactivity. Importantly, women show significantly greater methylation across the GR promoter exon 1F compared to men. The averaged methylation levels and each of the two polymorphisms are highly significant independent predictors of total cortisol response (AUC) in the TSST. These findings have important implications for understanding the molecular mechanisms underlying gender differences in stressrelated mental health disorders, and underscore the unique value of modeling both epigenetic and genetic information in conferring vulnerability to stress.