The epicardial release of immunoreactive atrial natriuretic peptides (ir-ANPs) in inside-out perfused rabbit atria has been reported. In order to determine the presence of ir-ANPs in pericardial fluid and to evaluate their biochemical characteristics, we measured the concentration of ir-ANPs in pericardial fluid obtained from the patients with congenital heart diseases during open heart surgery. Serial dilution curves made with the extrats of pericardial fluid using Sep-Pak C18 cartridges were parallel with standard curve. The concentration of ir-ANPs in pericardial fluid was significantly lower than the corresponding plasma concentration. On gel permeation and reverse-phase high performance liquid chromatography, the ir-ANPs in pericardial fluid, plasma and atrial appendage showed both high and low molecular weights. The major peak of ir-ANPs in plasma was observed at the corresponding fraction to the alpha-human ANP and considerable amount of high molecular weight form of ir-ANPs was observed in pericardial fluid. However, the major peak of ir-ANPs in atrial appendage was observed at the corresponding fraction to the rat pro-ANP. The data suggest that ir-ANPs exist both high and low molecular weight forms in pericardial fluid.