We have previously shown that extracellular fluid (ECF) is translocated by atrial contraction. Following on from this finding we have proposed a two-step sequential mechanism for the regulation of stretch-activated secretion of atrial natriuretic peptide (ANP): myocytic release of ANP into the surrounding paracellular space followed by the translocation of ECF with the released ANP into the bloodstream. This latter step is induced by atrial contraction, and is therefore controlled by atrial workload. However, the mechanism that regulates the changes in translocation of the ECF has not been defined. To define the relationship between the atrial workload, ECF translocation, size of the extracellular space (ECS) and ANP secretion, experiments have been performed in isolated perfused beating rabbit atria. Atrial workload, transendocardial translocation of the ECF and the secretion of ANP were quantified. Changes in the size of the atrium and the ECS were determined by a newly developed methodology in the beating atria. Atrial workload determined the translocation of the ECF and released ANP with waning of the translocation at higher myocardial workloads. Atrial workload inversely determined the size of the atrium and the ECS. The latter directly determined the translocation of the ECF in terms of atrial workload. From these data we suggest that the size of the ECS is an intimate modulator of the translocation of the ECF and released ANP, and that the phenomenon of waning of the transendocardial translocation that appeared at higher atrial workloads is closely related to the shrinkage of the ECS. Experimental Physiology (2002) 87.2, 185-194.