Atrial myocytes cultured for 7 days in serum-free medium secrete a 15K form of atrial natriuretic peptide (15K ANP), but isolated perfused rat hearts secrete the major circulating form of the hormone, a 3K peptide, 3K ANP. This difference was examined in the present study. 15K ANP was purified from rat atria, and sequencing analysis demonstrated that this atrial-derived ANP possessed an NH2-terminal sequence identical to that of pro-ANP; this is consistent with other reports suggesting that the major form of ANP in the atria is ANP-(1-126). Fresh rat serum was shown to cleave efficiently ANP-(1-126) to form a 3K immunoactive ANP-related peptide. Upon purification and sequencing the identity of this peptide was confirmed as ANP-(99-126); ANP-(99-126) was relatively resistant to further proteolysis by rat serum. To probe further the specificity of the serum conversion, synthetic ANP-(92-126) was used as a substrate; purification and sequencing of the immunoactive product peptide verified its identity as ANP-(99-126). Since purified thrombin and plasma kallikrein both cleaved ANP-(1-126) to 3K ANP-like material, inhibitors of these enzymes were tested for their ability to inhibit the serum cleavage of ANP-(1-126). D-Phe-Phe-Arg-Chloromethylketone (D-Phe-Phe-Arg-CMK) and D-Phe-Pro-Arg-CMK both inhibited serum ANP cleavage by over 90% at low micromolar concentration. When these inhibitors were added to the isolated heart perfusate, 3K ANP was still released by the atria, indicating that ANP processing occurs in the heart in a region not accessible to the inhibitors (i.e. intracellularly) or that the ANP-processing enzyme(s) is not inhibited by these CMK analogs and is, therefore, not related to serum-derived proteases.