Energy-dependent subcellular Ca2+ sequestration was studied in the presence of ruthenium red using rat pancreatic acini, which had been permeabilized by exposure to medium nominally free of Ca2+. The initial rate of Ca2+ uptake (approximately 2,800 pmol.min-1.mg acinar protein-1) quickly slowed, and a mean steady-state Ca2+ content of approximately 3,000 pmol/mg was reached after 5-10 min of incubation at 37 degrees C. Ca2+ uptake was stimulated by submicromolar Ca2+ concentrations (K0.5 = 156 nM); required Mg2+-ATP (K0.5 = 0.78 mM) was greatest at a pH of 7.0 and was abolished by the Ca2+ ionophore A23187. Other nucleotide phosphates as well as p-nitrophenylphosphate were relatively poor substrates, supporting Ca2+ uptake at initial rates that were 6-14% of those measured in the presence of ATP. These results show that pancreatic acini permeabilized without detergents possess a nonmitochondrial Ca2+ transporting system not located in the plasma membrane but with the properties expected of a major regulator of acinar cytosolic Ca2+ concentration.