Permeabilized rabbit pancreatic acinar cells were used to study the effects of Ca2+ pump inhibition and Ca2+ store depletion on the sensitivity of internal Ca2+ stores to emptying by inositol 1,4,5-trisphosphate (Ins-1,4,5-P3). Complete inhibition of pump activity by thapsigargin resulted in the monoexponential loss of 92% of the actively stored Ca2+ with a half-time of 6.2 min. Under these conditions, Ca2+ release evoked by a submaximal concentration of Ins-1,4,5-P3 did not cease after 1.5 min, as was observed in the absence of thapsigargin, but continued for at least 5 min. This observation suggests that under normal conditions of Ca2+ pumping, a substantial part of the internal Ca2+ stores is not depleted by the action of Ins-1,4,5-P3 due to compensatory Ca2+ uptake. Evidence in support of the idea of compensatory Ca2+ pumping was obtained in exchange experiments performed in the absence of thapsigargin. The slow kinetics of sustained Ca2+ release in the absence of Ca2+ pump activity suggests that Ca2+ is released from stores containing either relatively few or less sensitive Ins-1,4,5-P3-operated Ca2+ release channels. Gradual emptying of the internal Ca2+ stores by thapsigargin did not affect the potency with which Ins-1,4,5-P3 released Ca2+, indicating that the intravesicular Ca2+ content does not control the sensitivity of the Ins-1,4,5-P3-operated Ca2+ channel to activation by Ins-1,4,5-P3. This was confirmed using ruthenium red, which preferentially depleted the Ins-1,4,5-P3-releasable store without affecting the EC50 for Ins-1,4,5-P3-stimulated Ca2+ release. The data presented indicate that the quantal type of Ca2+ release observed with Ins-1,4,5-P3 requires compensatory Ca2+ pumping. Moreover, they support the idea that internal Ca2+ stores display differential sensitivities toward Ins-1,4,5-P3 rather than responding uniformly to this internal Ca(2+)-mobilizing messenger.