Ventricular myocytes were isolated from adult rat hearts using the technique of Wittenberg and Robinson (Cell Tissue Res. 216: 231-251, 1981). These cells exhibited morphology, input resistance, time constant, and excitability expected for cells in intact cardiac tissue. Pairs of these cells were electronically coupled, and junctional conductance was unaffected by transjunctional potential or hyperpolarization of both cells. Brief exposure of cell pairs to medium equilibrated with 100% CO2 or containing 0.1 mM octanol quickly and reversibly decreased junctional conductance. We conclude that gap junctions between pairs of ventricular myocytes possess physiological properties like those of junctions in many other tissues. This preparation will be useful in evaluating drug action on junctional communication in heart.