To investigate the mechanism by which an increase in pacing frequency or distension increases the secretion of atrial natriuretic peptide (ANP), the changes in atrial volume during contraction (atrial stroke volume), transmural transport of the extracellular fluid (ECF), and ANP secretion were quantified in the beating perfused rabbit atria. The atrium was stimulated by transmural field stimulation or by atrial distension induced by an increase in intraatrial pressure. Atrial stretch and incremental increases in pacing frequency up to 2 Hz activated the secretion of ANP coincident with an increase in atrial stroke volume and the transendocardial translocation of the ECF. These results showed positive relationships between changes in the secretion of ANP and the atrial stroke volume or the translocation of the ECF. The translocation of the ECF was also positively correlated with the change in atrial stroke volume. The accentuated secretion of ANP and translocation of the ECF waned at higher stimulating rates to show a peak value. Even under this condition, the secretion of ANP was a function of the translocation of the ECF. These data suggest that the increases in atrial stroke volume and translocation of ECF are fundamental factors in the ANP stimulation in response to atrial stretch and increases in atrial rate.