The mode of action of estrogen on β-adrenoceptor-mediated relaxation was investigated by using isolated ring preparations of thoracic aorta from ovariectomized rats. Administration of 17β-estradiol to ovariectomized rats significantly suppressed isoprenaline-induced relaxation of aortic rings. There was no alteration in the β-adrenoceptor binding characteristics. The suppressing action of 17β-estradiol on the N G -nitro-l-arginine and indomethacin-resistant relaxation induced by isoprenaline disappeared after pretreatment with N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525A), an inhibitor of cytochrome P450 (CYP). The levels of CYP2C11 expression were the highest of the CYP mRNAs examined in rat aorta. 17β-Estradiol replacement increased the expression of CYP2C11 mRNA in the aorta, compared with that in ovariectomized rats. These results suggest that estrogen suppresses β-adrenoceptor-mediated vasorelaxation, and that the mechanisms may be associated with alterations in CYP2C11 metabolites.