It has been suggested in this laboratory that the principal stimulus for the secretion of atrial natriuretic peptide (ANP) is the reduction of atrial distension and that the secretion of ANP is dependent on both atrial reduction volume and reduction frequency. To investigate the relationship among the changes in atrial pressure, distension, pacing frequency, and ANP secretion, we performed a series of experiments in the isolated perfused rabbit atria. Increase in atrial pressure without changes in transmural pressure and thus without volume changes did not raise immunoreactive ANP (irANP) secretion. Atrial distension without changes in intracavitary atrial pressure increased irANP secretion with the reduction. Electrical stimulation with atrial distension resulted in an increase in irANP secretion in proportion to pacing frequency. Incremental response of irANP secretion to electrical stimulation was accentuated by increasing atrial distension. Neither atrial pacing without distension nor distension without pacing raised irANP secretion. These results suggest that the direct and principal stimulus for irANP secretion in response to atrial pacing and distension is the length shortening of atrial myocytes and that the incremental response of irANP secretion to increasing pacing frequency is the result of an increase in frequency of the length shortening of atrial myocytes.