Selenium is an antioxidant trace element linked to cardiovascular disease and cancer. Although diet is a major source, relatively little else is known about independent determinants of selenium levels in free-living humans. In this study, we aimed to investigate the independent demographic, lifestyle, and dietary determinants of selenium levels in 1,997 men and 1,905 women in two large prospective U.S. cohorts. Toenail selenium levels were quantified using neutron activation analysis. Diet, geographic residence, demographic, and environmental factors were assessed by validated self-administered questionnaires. Multivariate generalized linear models were conducted to assess the independent relations of these factors with toenail selenium levels, correcting for measurement error in the diet. In multivariable-adjusted analyses, independent predictors of higher selenium were male gender (6.3% higher levels); living in West and Northern-Midwest U.S. regions (8.9% and 7.4% higher than Southern-Midwest regions, respectively); consumption of beef and bread products (between 0.7 - 2.5% higher per daily serving); and selenium supplement use (6.9% higher than non-users); whereas cigarette smoking (5-10% lower than never smokers) , older age (0.6% lower per 5 years), and consumption of eggs, white rice, dairy products, coffee, and alcohol (between 0.1 to 2.0% lower per daily serving) were associated with lower selenium. Multiple dietary and non-dietary factors independently predicted selenium levels, suggesting that both consumption and non-dietary processes (e.g., related to oxidant status) may affect levels. Significant geographic variation in selenium levels exists in the US.