Women, before menopause, are known to be resistant to the development of acute ischemic heart disease (AIHD). As the inhibition of platelet aggregation is reported to prevent incidences of AIHD, the effects of estradiol and estriol on ADP-induced platelet aggregation in platelet-rich plasma were determined. It was found that it was not estradiol, the most potent estrogenic hormone, but estriol, less potent than estradiol, that had a minimum inhibitory concentration (MIC) of 0.6 nmol/l for 100% inhibition of ADP-induced platelet aggregation. In contrast, the MIC of estradiol was 2.0 nmol/l (P<0.005, n=40). The stimulation of nitric oxide (NO) by 0.6 nmol/l estriol in platelet-rich plasma was 0.55 nmol/108 cells/h and the stimulation by the 2.0 nmol/l estradiol was 0.179 nmol/108 cells/h. Treatment of intact platelets with 0.05% Triton X-100 released a membrane NO synthase in the supernatant that had basal Km of 5.28 mmol/l with Vmax of 0.029 nmol NO/mg/h. The treatment of the supernatant with 0.6 nmol/l estriol decreased the Km to 3.42 mmol/l with increased Vmax to 0.337 nmol NO/mg/h. These results showed that estriol was one of the most potent inhibitors of platelet aggregation with MIC that was in subnanomolar ranges, which is lower than any other inhibitors currently known and suggested that estriol might prevent AIHD in women before menopause.