Our objective was to evaluate the role of vacuolar H+-ATPase and proton gradients in the refilling of Ca2+ stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca2+ stores were replenished with a Ca2+-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca2+ pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H+-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca2+](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca2+ stores which are differently operated by vacuolar proton ATPase.