Atrial natriuretic peptide (ANP) is a hormone with an important role in the regulation of extracellular fluid volume. The ANP gene is not only expressed in the heart, but the high concentration of ANP in cardiac blood makes it difficult to demonstrate extraatrial hormonal secretion in vivo. This issue was addressed during complete cardiopulmonary bypass (CPS) in 13 patients undergoing elective cardiac surgery in whom ANP concentrations were followed in the internal jugular vein, representing largely brain venous outflow, as well as a peripheral vein and radial artery, after the heart and lungs were excluded from circulation. Plasma ANP levels in the peripheral venous circulation showed no significant changes during extracorporeal circulation, although they tended to decrease (from 6.75 ± 2.16 fmol/mL to 4.76 ± 0.69 fmol/mL; P > 0.05). ANP levels in the radial artery decreased significantly after the exclusion of the heart (from 16.84 ± 3.51 fmol/mL to 6.83 ± 0.97 fmol/mL; P < 0.01). In contrast, ANP concentration in the internal jugular vein increased in 12 of 13 patients during the first 15 minutes of CPS (from 9.49 ± 1.96 fmol/mL to 15.96 ± 2.8 fmol/mL; P < 0.01) and remained above the levels found simultaneously in other sampling sites during CPS. High-performance liquid chromatography analysis of plasma extracts showed multiple peaks of ANP, but the elution patterns of peripheral venous blood and brain outflow were similar. One of the immunoreactive peaks was located at the position of standard human ANP (Ser99Met110-Tyr126). The isolated peptide, corresponding to circulating hormone, displaced 125I ANP from rat glomerular receptors, suggesting the possible biologic activity of the ANP isolated from brain venous outflow. Higher ANP levels in venous outflow from the head than elsewhere in the circulation could result from secretion of ANP with receptor-binding properties by the central nervous system, possibly as a compensatory response to the absence of the principal source of this hormone.