Objective: Postmenopausal estradiol (E₂) levels vary widely between individuals and this variation is an important determinant of diseases such as osteoporosis. It has been suggested that the estrogen receptor alpha (ESR1) gene may influence peripheral E₂ levels, but the role of common sequence variations in the ESR1 gene is unclear. Methods: In 631 postmenopausal women and 528 men from the Rotterdam Study, a population-based, prospective cohort study of individuals aged 55 years and over, ESR1 PvuII-XbaI haplotypes were determined and correlated with plasma E2 levels. Results: In women, haplotype 1 (T-A) was significantly associated with an allele-dose-dependent decrease in E₂. After adjusting for age, body mass index, years since menopause and testosterone levels, plasma E₂ levels decreased by 1.90 pmol/l per allele copy of this haplotype (P < 0.05). Extreme genotypes, representing 23 and 27% of the population, varied by 3.93 pmol/l. No association with plasma testosterone was observed. In a subset of 446 women, no association of genotype with plasma concentrations of dehydroepiandrosterone sulfate, androstenedione or estrone was seen. In men, none of the sex hormone levels was associated with the ESR1 PvuII-XbaI haplotypes. Conclusion: We have demonstrated a role for genetic variations in the ESR1 gene in determining post-menopausal E₂ levels in women.