The distribution of adrenergic receptors in specific components of the heart such as vessels and myocytes cannot be determined easily with assays of membranes prepared from homogenates of whole tissue. Accordingly, we characterized the binding of the potent nonsubtype selective antagonist [125iodo]cyanopindolol to β-receptors in unfixed transmural slices of feline and canine left ventricle. Specific binding ratios >90% were achieved at radioligand concentrations near Kd and >80% at saturating ligand concentrations. Binding of radioligand to receptors in transmural slices was rapid, saturable, stereoselective, and displaceable by antagonists and agonists with the rank order of potency expected of β-adrenergic receptors. Analysis of binding isotherms indicated maximum binding capacities of 27.8 ± 6.6 and 40.6 ± 5.1 fmol/mg tissue protein and dissociation constants of 10.1 ± 1.8 and 21.3 ± 1.6 pM in feline and canine ventricular slices, respectively. The distribution of β-receptors in myocytes and selected vascular components of the heart was determined with quantitative film autoradiography and high resolution computer-based analysis and display of the density of binding sites, maximum binding capacity, and binding affinity measurements. The results of autoradiographic analysis revealed a uniform transmural distribution of receptors in regions composed primarily of ventricular myocytes but an inverse relation between the density of β-receptors and the diameter of coronary vessels. Large epicardial conductance arteries had half the receptor density of subjacent myocytes; small mural arteries had approximately 60% of the β-receptor density of nearby myocytes, and the coronary resistance arterioles had the highest receptor density of any vascular compartment, which was equivalent to that of myocytes. The methods developed should be of particular value in characterizing the distribution and function of receptor subtypes and mechanisms of regulation of adrenergic responsiveness in intact myocardium.