To determine the role of endothelial atriopeptin (AP) receptors, we examined the effect of AP-III on the morphology and macromolecular permeability of monolayer cultures of bovine aortic endothelial cells. AP-III alone (10(-9)-10(-6) M) had no observable effect on the morphology of the monolayers or their permeability to 125I-labeled albumin. However, incubation of the endothelial monolayers with AP-III (10(-8)-10(-6) M) antagonized thrombin-induced (1 unit/ml) cell-shape change and the formation of intercellular gaps. AP-III also opposed the effect of thrombin on the distribution of actin filaments in the endothelial cytoskeleton. Further, thrombin caused a 2-fold increase in monolayer permeability to 125I-labeled albumin, which was abolished by 10(-8)-10(-6) M AP-III pretreatment. Taken together with the findings that AP-III exhibited specific and saturable binding in these cells, these data suggest that AP regulates endothelial permeability through a receptor-mediated process.