Abstract-To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter Ϸ300 m) obtained from 42 heart valve donors (20 men and 22 women; age range, 3 to 65 years; mean age, 46 years) were mounted in Mulvany myographs. In the presence of the cyclooxygenase inhibitor indomethacin, bradykinin relaxed preconstricted HCMAs in a concentration-dependent manner. N G -nitro-L-arginine methyl ester and ODQ (inhibitors of nitric oxide [NO] synthase and guanylyl cyclase, respectively) and the NO scavenger hydroxocobalamin, either alone or in combination, shifted the bradykinin concentration-response curve to the right. Removal of H 2 O 2 (with catalase), inhibition of cytochrome P450 epoxygenase (with sulfaphenazole or clotrimazole) or gap junctions (with 18␣-glycyrrhetinic acid or carbenoxolone), and blockade of large-(BK Ca ) and small-(SK Ca ) conductance Ca 2ϩ -dependent K ϩ channels (with iberiotoxin and apamin), either alone or in addition to hydroxocobalamin, did not affect bradykinin. In contrast, complete blockade of bradykinin-induced relaxation was obtained when we combined the nonselective BK Ca and intermediate-conductance (IK Ca ) Ca 2ϩ -dependent K ϩ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K ϩ channels (K IR ) and Na ϩ /K ϩ -ATPase (with BaCl 2 and ouabain, respectively) shifted the bradykinin concentration-response curve 10-fold to the right but did not exert an additional effect in the presence of hydroxocobalamin. In conclusion, bradykinin-induced relaxation in HCMAs depends on (1) the activation of guanylyl cyclase, K IR , and Na ϩ /K ϩ -ATPase by NO and (2) IK Ca and SK Ca channels. The latter are activated by a factor other than NO. This factor is not a cytochrome P450 epoxygenase product or H 2 O 2 , nor does it depend on gap junctions or BK Ca channels. Key Words: bradykinin Ⅲ arteries Ⅲ endothelium-derived factors Ⅲ nitric oxide E ndothelium-dependent relaxation induced by bradykinin cannot fully be attributed to the release of nitric oxide (NO). In resistance-size vessels, a large proportion of endothelium-derived relaxation involves the release of endothelium-derived hyperpolarizing factors (EDHFs). 1 Putative EDHF candidates are prostacyclin, S-nitrosothiols, K ϩ , cytochrome P450 products of arachidonic acid (epoxyeicosatrienoic acids [EETs]), and H 2 O 2 , 2-8 and EDHF-dependent responses have been reported to involve large-, intermediate-, and/or small-conductance Ca 2ϩ -activated K ϩ channels (BK Ca , IK Ca , and SK Ca, respectively), inwardly rectifying K ϩ (K IR ) channels, Na ϩ /K ϩ -ATPase, and gap junctions. 4,5,8 -10 Busse et al 9 recently summarized all currently available data on EDHF and proposed that EDHF-mediated relaxation (ie, relaxation observed in the absence of NO) depends on the activation of endothelial IK Ca and SK Ca channels. 4 Such activation results in the release of K ϩ into the myoendothelial spac...