Puerarin is the main isoflavone found in Pueraria lobata (Willd) Ohwi, which has been used in therapy for various cardiovascular diseases. The present study examined the effects of puerarin on the large-conductance voltage- and Ca2+-activated potassium (BK(Ca)) channel and on rat thoracic aortas. BK(Ca) channels encoded with either alpha (BK-alpha) or alpha/beta subunits (BK-alpha+beta1) were heterologously expressed in Xenopus oocytes or human embryonic kidney 293 cells. The activities of BK(Ca) channels were measured using excised patch-clamp recordings. Puerarin activated BK-alpha+beta1 currents with a half-maximal concentration (EC50) of 0.8 nM and a Hill coefficient of 1.11 at 10 microM Ca2+ and with an EC50 of 12.6 nM and a Hill coefficient of 1.08 at 0 microM Ca2+. Puerarin (1 nM) induced a 16-mV leftward shift in the conductance-voltage curve for BK-alpha+beta1 currents at 10 microM Ca2+ and at 100 nM induced a 26-mV leftward shift at 0 microM Ca2+. Puerarin mainly increased the BK-alpha+beta1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of the beta1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BK(Ca) channels with weaker potency. In addition, puerarin (0.1 to 1000 microM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1 microM noradrenaline bitartrate (EC50 = 1.1 microM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BK(Ca) channels. This is the first study demonstrating that puerarin activates BK(Ca) channels, especially BK-alpha+beta1 channels. The activation of the BK(Ca) channel probably contributes to the puerarin-mediated vasodilation action.