The myocardial interstitium is important in regulating cardiac function. Between the atrial lumen and the pericardial space are transmural pathways, and movement of interstitial fluid (ISF) through these pathways is one of the main driving forces regulating translocation of substances from the interstitium into the blood. To define how ISF translocation from the interstitial space into the luminal space is regulated by each component of atrial hemodynamics, we devised a new rabbit atrial model in which each physical parameter could be controlled independently. Using this system, we also defined the physiological role of the cardiac Na⁺/Ca²⁺ exchanger on secretion of atrial natriuretic peptide (ANP) by depletion of extracellular Na⁺ ([Na⁺]_o). Increases in stroke volume and atrial end-systolic volume increased ISF translocation and ANP secretion. However, an increase in atrial rate did not influence ISF translocation but, rather, increased ANP secretion. Gradual depletion of [Na⁺]_o caused gradual increases in ANP secretion and intracellular Ca²⁺ ([Ca²⁺]_i), which were blocked in the presence of Ca²⁺-free buffer and Ni²⁺, but not in the presence of KB-R7943, diltiazem, mibefradil, caffeine, or monensin. Amiloride and its analog blocked an increase in ANP secretion but not an increase in [Ca²⁺]_i by [Na⁺]_o depletion. Therefore, we suggest that ANP secretion and ISF translocation may be differently controlled by each physical factor. These results also suggest that the increase in ANP secretion in response to [Na⁺]_o depletion may involve inhibition of Na⁺/Ca²⁺ and Na⁺/H⁺ exchangers but not an increase in [Ca²⁺]_i.