Objectives: I _Ks, the slow component of the delayed rectifier potassium current, underlies a strong β-adrenergic regulation in the heart. Catecholamines, like isoproterenol, induce a strong increase in I_Ks. Recent work has pointed to an opposing biological effect of β₁- and β₃-adrenoceptors in the heart. However the role of these subtypes in the regulation of cardiac ion channel function is unknown. Methods: We investigated the effects of β₁- and β₃-adrenoceptor modulation on I_Ks in guinea-pig ventricular myocytes, using patch-clamp techniques. Results: Superfusion with 100 nmol/l isoproterenol increased the step current amplitude by 81.3±8.0%. In contrast, after block of β₁- (1 μmol/l atenolol) and β₂-receptors (1 μmol/l ICI118,551), isoproterenol induced a reduction of the step current amplitude by 34.3±3.5%. The β₃-selective agonist BRL37344 significantly reduced the I_Ks step current at +70 mV in a concentration-dependent manner (IC₅₀: 5.01 nmol/l). In the presence of bupranolol (β₁-, β₂- and β₃-adrenoceptor antagonist), the effect of BRL37344 was markedly attenuated, from 27.3±5.6% (100 nmol/l BRL37344 alone) to 4.0±1.3% (100 nmol/l BRL37344+1 μmol/l bupranolol). BRL37344 (100 μmol/) did not alter current amplitudes of KvLQT1/minK expressed in CHO cells or in Xenopus oocytes, excluding a direct effect of BRL37344 on the channel. 1 μmol/l BRL37344 mildly prolonged action potentials in guinea pig ventricle (APD₉₀:+7.8%) Conclusions: We have demonstrated a functional coupling between the β₃-adrenoceptor and ion channel function in the mammalian heart. Our findings point to a potential role for β₃-adrenoceptors in cardiac electrophysiology and pathophysiology.