Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40–/–mice, the PR interval was prolonged (47.4±1.4 in wild-type [WT] [n=6] and 57.5±2.8 in Cx40–/–[n=6];P<0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40–/–hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40–/–mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7±0.7 ms in WT [n=6] and 6.5±0.7 ms in Cx40–/–[n=6];P<0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40–/–mice (74.5±3 cm/s in WT [n=7] and 43.7±6 cm/s in Cx40–/–[n=7];P<0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40–/–RBB activation time with respect to P time (P-RBB time; 41.6±1.9 ms in WT [n=7] and 55.1±1.3 ms in [n=7];P<0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40–/–mice and provide new insight into the role of gap junctions in cardiac impulse propagation.