The cellular mechanisms by which mechanical forces regulate myocardial function such as secretion of atrial natriuretic peptide (ANP), are uncertain. We studied the effects of thapsigargin, a specific inhibitor of sarcoplasmic reticulum Ca2+ adenosine triphosphatase, that depletes intracellular Ca2+ stores, on basal and atrial stretch-induced ANP secretion in the isolated, perfused, paced rat heart preparation. Addition of 300 nM thapsigargin into the perfusate caused gradual increase in perfusion pressure, contractile force and ANP release (P < 0.001). Thapsigargin pretreatment at concentrations (30 and 100 nM) that did not affect baseline cardiac function or hormone secretion blocked mechanical stretch-induced increase in ANP secretion. These results suggest that thapsigargin-sensitive intracellular Ca2+ pools serve as mechanotransducers in the mechanical loading-induced changes in cardiac myocytes.