Rabbit pancreatic acinar cells, permeabilized by saponin treatment, were used to study the kinetics of ATP-dependent Ca2+ uptake and release in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3)-sensitive and -insensitive stores. Permeabilized acinar cells rapidly accumulated Ca2+ to steady-state. At steady state, approximately 60% of actively stored Ca2+ resided in the Ins-1,4,5-P3-sensitive store. Kinetic analysis of the Ca2+ uptake process revealed that the initial Ca2+ uptake rate was 1.7 times higher in the Ins-1,4,5-P3-insensitive store as compared to the Ins-1,4,5-P3-sensitive store. On the other hand, the Ca2+ uptake capacity was 1.6 times higher in the Ins-1,4,5-P3-sensitive store as compared to the Ins-1,4,5-P3-insensitive store. The Ca2+ uptake rate in the Ins-1,4,5-P3-sensitive store remained virtually constant for at least 4 min, whereas in the Ins-1,4,5-P3-insensitive Ca2+ store this rate progressively declined with time. These observations are compatible with: (i) an Ins-1,4,5-P3-sensitive store containing relatively few Ca2+ pumps but possessing a relatively high Ca2+ uptake capacity, which may reflect the presence of a substantial amount of Ca2+ binding protein; and (ii) an Ins-1,4,5-P3-insensitive Ca2+ store containing relatively many Ca2+ pumps but possessing a relatively low Ca2+ uptake capacity, which may reflect the presence of little if any Ca2+ binding protein. The data presented are consistent with the idea of a heterogeneous distribution of Ca2+ pumps, Ca2+ binding proteins and Ca2+ release channels between intracellular Ca2+ storage organelles.