The endocardial to epicardial gradient in action potential duration (APD) is responsible for the normal pattern of repolarization in the heart of many mammals including man. Differences in repolarizing K⁺ currents contribute to the gradient in APD. In the present study we investigate the role of Ga11-coupled receptors in the generation of regional differences in APD and repolarizing K⁺ currents. Single myocytes from endocardial and epicardial regions of the left ventricle were isolated from wild type (wt) controls and Ga11-deficient (gna11^-/-) mice and investigated using the whole-cell patch-clamp technique. In endocardial myocytes from gna11^-/-mice, APD₉₀ was significantly shorter compared to wt (39.7±3.7 ms, n=25 vs. 80.7 ± 7.7 ms, n=32, p<0.001) and indistinguishable from the APD₉₀ in epicardial myocytes (31.1±2.8ms, n=34, n.s.). No change was observed in epicardial myocytes. The fast component of I_to measured at +40 mV was increased to a larger extent in endocardial (24.2±3.3 pApF^-1, n=22 vs. 14.1±1.3 pApF^-1, n=17, p<0.05) than in epicardial myocytes (41.4±1.9 pApF^-1, n=34 vs. 32.9±2 pApF^-1, n=31, p<0.05) of gna11^-/-mice. In ECG recordings of gna11^-/mice the direction of the mean QRS-vector was unchanged, whereas the direction of the T-vector was shifted leftwards. These findings suggest, that Ga11-coupled receptors play a major role in the signaling cascade responsible for regional differences of I_to magnitude and hence APD.