The rapidly activating delayed rectifier potassium current (I-Kr) plays a critical role in cardiac repolarization. Although I-Kr is known to be regulated by both alpha(1)- and beta(1)-adrenergic receptors (ARs), the cross-talk and feedback mechanisms that dictate its response to alpha(1)- and beta(1)-AR activation are not known. In the present study, I-Kr was recorded using the whole-cell patch-clamp technique. I-Kr amplitude was measured before and after the sequential application of selective adrenergic agonists targeting alpha(1)- and alpha(1)-ARs. Stimulation of either receptor alone (alpha(1)-ARs using 1 M phenylephrine (PE) or beta(1)-ARs using 10 mu M xamoterol (Xamo)) reduced I-Kr by 0.22 +/- 0.03 and 0.28 +/- 0.01, respectively. The voltage-dependent activation curve of I-Kr shifted in the negative direction. The half-maximal activation voltage (V-0.5) was altered by -6.35 +/- 1.53 and -1.95 +/- 2.22 mV, respectively, with no major change in the slope factor (k). When myocytes were pretreated with Xamo, PE-induced reduction in I-Kr was markedly blunted and the corresponding change in V-0.5 was significantly altered. Similarly, when cells were pretreated with PE, Xamo-induced reduction of I-Kr was significantly attenuated. The present results demonstrate that functional cross-talk between alpha(1)- and beta(1)-AR signaling regulates I-Kr. Such non-linear regulation may form a protective mechanism under excessive adrenergic stimulation.