In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca²⁺-ATPase thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca²⁺-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca²⁺-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca²⁺-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca²⁺-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.