Abstract--To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter 300 µm) 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 H₂O₂ (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 Ca²⁺-dependent K⁺ 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) Ca²⁺-dependent K⁺ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K⁺ channels (K_IR) and Na⁺/K⁺-ATPase (with BaCl₂ 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⁺/K⁺-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 H₂O₂, nor does it depend on gap junctions or BK_Ca channels.