The β-adrenergic agonist isoproterenol stimulates immunoreactive atrial natriuretic peptide (IR-ANP) secretion by superfused rat atria in vitro. β-Adrenergic agonists alter the cellular handling of calcium, which culminates in a rise in the systolic calcium concentration. This is achieved by increasing calcium influx through voltage-dependent calcium channels and by increasing the storage pool of calcium in the sarcoplasmic retlculum (SR). We therefore asked the question whether isoproterenol-stimulated IR-ANP secretion was dependent on the protein kinase A-induced rise in systolic calcium or was due to a direct effect of protein kinase A activation. Isolated rat left atria paced at 3 Hz were superfused in vitro. IR-ANP secretion was determined by radioimmunoassay of timed collections of the superfusate. Superfusion with 0.1 μM isoproterenol or 0.5 mM dibutyryl cyclic AMP increased IR-ANP secretion twofold. Stimulated IR-ANP secretion was lowered to near baseline by lowering the buffer calcium concentration from 1.8 to 0.2 mM or by adding to the superfusate 10 μM nitrendipine (a calcium-channel blocker) or 1 μM ryanodine (an inhibitor of SR calcium release). Superfusion of nonbeating, electrically quiescent left atria with 0.1 μM isoproterenol failed to stimulate IR-ANP secretion. We conclude: 1) Isoproterenol-stimulated IR-ANP secretion is dependent on calcium influx through voltage-dependent calcium channels and on the release of calcium from the SR. 2) Enhanced calcium influx alone is not adequate to maintain isoproterenol-stimulated IR-ANP secretion. 3) The SR appears to be the primary source of calcium for isoproterenolstimulated IR-ANP secretion. 4) The stimulatory effect of isoproterenol on IR-ANP secretion is dependent on electrical membrane activity. 5) Thus, protein kinase A activation does not appear to have a direct effect on IR-ANP secretion. Its effect appears to be mediated by protein kinase A-directed changes in the handling of calcium by atrial cardiocytes. These results suggest that calcium-channel blockers may lower plasma ANP levels in man when sympathetic tone is high. Lowering plasma ANP may be responsible, in part, for fluid retention in patients treated with calcium-channel blockers.