Gap junction channels consisting of connexin protein mediate electrical coupling between cardiac cells. Expression of two connexins, connexin40 (C×40) and connexin43 (C×43), has been studied in ventricular myocytes from normal and hypertensive rats. Polyclonal affinity-purified rabbit antibodies to C×43 and C×40 have been used for immunohistochemical analysis on frozen sections from rat heart. These studies revealed coexpression of C×43 and C×40 in ventricular myocytes. In addition, C×40 is preferentially expressed in three distinct regions: first, in the endothelial layer of the heart blood vessels but not in the smooth muscle layer of the arteries; second, in the ventricular conductive myocardium, particularly in the atrioventricular bundle and bundle branches, where C×43 is not observed; and third, in the myocyte layers close to the ventricular cavities. These results suggest that C×40 is preferentially expressed in the fast conducting areas of myocardial tissue. Expression of both C×40 and C×43 was also found in immunoblots from normal and hypertensive rat myocardiocytes. Under hypertensive conditions (ie, in spontaneous hypertensive rats and in transgenic rats that exhibit hypertension due to expression of an exogenous renin gene), we found a 3.1-fold increase in C×40 expression, compared with normal myocardium. Furthermore, we detected a 3.3-fold decrease in C×43 protein level in transgenic hypertensive rats. The coexpression of C×40 and C×43 proteins in rat myocytes, their spatial distribution, and the increased amount of C×40 protein during cardiac hypertrophy suggest that C×40 may be involved in mediating fast conduction under normal and pathological conditions. The increased expression of C×40 in hypertrophic heart may be a compensatory mechanism to increase conduction velocity.