Ventricular arrhythmias are frequently observed in the elderly population secondary to alterations of electrophysiological properties which occur with the normal aging process of the heart. However, the underlying mechanisms remain poorly understood. The aim of the present study was to determine specific age-related changes in electrophysiological properties and myocardial structure in the ventricles which can be related to a structural-functional arrhythmogenic substrate. Multiple unipolar electrograms were recorded in vivo on the anterior ventricular surface of 4 control and 7 aged rats, during normal sinus rhythm and ventricular pacing. Electrical data were related to morphometric and immunohistochemical parameters of the underlying ventricular myocardium. In aged hearts total ventricular activation time was significantly delayed (QRS duration:+69%) while ventricular conduction velocity did not change significantly, as compared to control hearts. Moreover, ventricular activation patterns displayed variable numbers of epicardial breakthrough points whose appearance could change with time. Morphological analysis in aged rats revealed that heart weight and myocyte transverse diameter increased significantly, scattered microfoci of interstitial fibrosis were mostly present in the ventricular subendocardium and gap junction connexin expression decreased significantly in ventricular myocardium, as compared to controls. Our results show that in aged hearts delayed total ventricular activation time and abnormal activation patterns are not due to delayed myocardial conduction and suggest the occurrence of impaired impulse propagation through the conduction system leading to uncoordinated myocardial excitation. Impaired interaction between conduction system and ventricular myocardium might create a potential reentry substrate, contributing to higher incidence of ventricular arrhythmias in the elderly population.