Atrial natriuretic peptide (ANP) is a hormone secreted predominantly by atrial myocytes. Although atrial distension is the primary stimulus of ANP secretion, several hormones have also been implicated in the regulation of ANP secretion. alpha-Thrombin, a serine protease participating in the blood coagulation system, has additional hormone-like effects in several cell types, apparently via interaction with specific cell surface receptors. Here we report that alpha-thrombin enhanced ANP secretion from isolated rat atrium within 10 min, in a concentration-dependent manner. The protease also significantly increased ANP release from cultured atrial myocytes, in a concentration-dependent manner. The alpha-thrombin-induced release of ANP from cultured atrial myocytes was completely abolished by hirudin, a specific alpha-thrombin protease inhibitor. Furthermore, synthetic peptides, identical in their amino acid sequence to the N-terminal segment of the proteolytically cleaved thrombin receptor, enhanced ANP release from adult rat cultured atrial myocytes. Our data suggest that thrombin may regulate ANP release from the cardiac atrium. This action involves activation of thrombin receptors in atrial myocytes.