This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl − channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736μl for volume load vs. 129μl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca 2+ -activated Cl − channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl − channel, diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl − channels may differently regulate stretch-activated ANP secretion.