The uptake of 45Ca2+ was studied in microsomes prepared from isolated mouse pancreatic acini. These microsomes accumulated 45Ca2+ in the presence of ATP; uptake was potentiated by addition of oxalate. Sequestered microsomal 45Ca2+ was only gradually removed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) but was readily released by the divalent cation ionophore A23187. Inhibitors of mitochondrial oxidation and mitochondrial calcium transport had little effect on microsomal 45Ca2+ uptake. A separate subcellular fraction enriched in plasma membranes took up 45Ca2+ poorly compared with the microsomal fraction. Half-maximal 45Ca2+ uptake by the microsomal fraction was observed at a free Ca2+ concentration of 1.1 microM. 45Ca2+ uptake was dependent on Mg-ATP and showed a pH optimum at 6.8-7.0. Subfractionation of the total microsomes into "heavy" and "light" microsomal fractions indicated higher 45Ca2+ uptake activity associated with the heavy fraction. A Ca2+-activated, Mg2+-dependent ATPase was demonstrated in this fraction. Stimulation of pancreatic acini with the cholecystokinin analogue caerulein prior to homogenization increased the subsequent rate of 45Ca2+ uptake by the microsomal fraction.