A reduction in fat intake has been associated with decreased estrogen levels in dietary intervention studies. However, previous cross-sectional studies conducted mainly among Western populations did not find a positive association between fat intake and postmenopausal estrogen levels. This study examined the cross-sectional association of fat intake with serum levels of estrone, estradiol, testosterone, and dehydroepiandrosterone sulfate (DHEAS) in women. Study subjects were 324 healthy postmenopausal Japanese women. Diet including fat intake was assessed by a validated semiquantitative FFQ. After controlling for age and other potential breast cancer risk factors, serum estrone was positively associated with the percentage of energy from total fat (P = 0.04). The associations of serum estrone with monounsaturated fat and polyunsaturated fat were of borderline significance (P = 0.05). Serum DHEAS was positively associated with the percentage of energy from total fat (P = 0.007), saturated fat (P = 0.009), monounsaturated fat (P = 0.006), and polyunsaturated fat (P = 0.04). Serum estrone and DHEAS concentrations increased 11.8 and 9.3%, respectively, with a 5% increase in the percentage of energy from total fat. These data suggest that a high intake of fat is associated with higher serum levels of estrone and DHEAS in postmenopausal women.