Iron overload, expressed as increased body iron stores, has been recognized as a potential hazard because it promotes the generation of oxygen radicals. We analyzed factors associated with serum ferritin levels (an indicator of body iron stores) among middle-aged women with a high prevalence of nutrient supplement use. Serum ferritin concentrations were determined on automated immunoassay for 487 healthy women with the mean age of 57 years who participated in the New York University Women's Health Study. The mean serum ferritin concentration in postmenopausal women was more than twice that in premenopausal women. Serum ferritin concentrations progressively increased with advancing age, but adjustment for menopausal status considerably weakened this association. Among non-dietary factors, nonwhite ethnicity, obesity and cigarette smoking were positively associated with serum ferritin concentrations. After adjustment for these factors and for menopausal status, serum ferritin levels were positively associated with meat intake and multivitamin use and inversely associated with breakfast cereal consumption. However, none of these lifestyle factors positively associated with serum ferritin levels had a significant impact on serum ferritin levels above 100 ng/ml (approximately equal to median concentration). Our results suggest that iron overload seems unlikely among middle aged women through their diet and nutritional supplements.