Atrial distension, which stimulates atrial natriuretic peptide secretion by atrial myocytes, also stretches nonmuscle cells. In a noncontracting in vitro preparation of combined right and left atria we demonstrated by electron microscopy that, at 37°C, transition from zero pressure to a physiological distending pressure of 5.1 mm Hg rapidly rendered atrial endocardial endothelium permeable to the macromolecular probes horseradish peroxidase (HRP;Mr, ≈40,000) and wheat germ agglutinin-HRP (Mr, ≈70,000); each probe was introduced at the atrial cavitary endocardial surface. Stretch-dependent permeabilization was also demonstrable in spontaneously contracting atria, was reversed by removing the distending pressure, and was unaffected by varying external Ca2+concentration from 0.2 to 1.4 mM or by experimental perturbations that markedly decrease ANP secretory rates. Although transendocardial HRP and wheat germ agglutinin-HRP passage required stretch, native ferritin (Mr, ≈500,000) could traverse unstretched endocardium. Probes were detected in noncoated endocardial vesicles and intercellular junctions between endocardial cells, but the relative contributions of vesicular transcytosis and paracellular diffusion could not be determined. Although HRP entered plasmalemmal caveolae of myocytes in stretched atria, myocytes did not internalize HRP by fluid-phase endocytosis. Distending pressure also caused apparent flow reversal in thebesian blood vessels, with retrograde transfer of HRP across the endocardium into the myocardium. HRP and ferritin presented at the external surface of the epicardium (visceral pericardium) were endocytosed by mesothelial cells, entered junctions between mesothelial cells, and readily crossed the epicardium of both stretched and unstretched preparations.