Background —Myocardial conduction depends on intercellular transfer of currrent at gap junctions. Atrial myocytes express three different gap junction channel proteins—connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40)—whereas ventricular myocytes express only Cx43 and Cx45. However, the physiological roles of individual connexins are unknown. We have previously shown that mice heterozygous for a null mutation in the gene encoding Cx43 (Cx43 ^+/− mice) express 50% of the normal amount of Cx43 in ventricular myocardium and exhibit marked slowing of ventricular conduction. Methods and Results —To determine whether atrial conduction is affected in Cx43 ^+/− mice, we measured atrial conduction velocity in isolated hearts, performed detailed ECG and electrophysiological studies in intact animals, and determined the amount of cardiac connexins in atrial and ventricular tissue. Ventricular conduction velocity was reduced by 38% in Cx43 ^+/− mice compared with wild-types, but atrial conduction velocity in the same hearts was normal. QRS duration was significantly greater in Cx43 ^+/− mice than in wild-types, but P-wave duration and amplitude did not differ. Atrial expression of Cx43 was reduced by 50%. Conclusions —These results indicate that Cx43 is a principal conductor of intercellular current in the ventricle because ventricular conduction is significantly slowed when Cx43 content is reduced by only 50%. In contrast, a similar reduction in Cx43 content in atrial muscle has no effect on atrial conduction, suggesting that Cx40 (which is expressed in atrial but not ventricular myocytes) is a major electrical coupling protein in atrial muscle. Thus, Cx43 and Cx40 may be chamber-specific determinants of myocardial conduction.