The distribution of binding sites for atrial natriuretic peptide in cardiac ventricles of several mammalian species, including rat and human, was determined by in vitro autoradiography. The results revealed a unique anatomic localization of atrial natriuretic peptide binding sites to endomural vessels (Thebesian vessels), which communicate directly with the ventricular chambers. Digital image analysis indicated that these vascular channels possessed binding site densities comparable to those of the renal glomeruli, a major target site for circulating atrial natriuretic peptide. In contrast, no specific labeling of branches of the coronary arteries and veins was detected. The discrete localization of atrial natriuretic peptide binding sites to this "primitive" cardiac circulatory system allows speculation as to the role of this hormone in the regulation of endocardial circulation during cardiac development, normal ventricular function, and in coronary insufficiency.