The distribution of nonmitochondrial Ca2+ pumping sites and the site of action of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) were studied in subcellular fractions of human neutrophils. In homogenates, two different Ca2+ pools could be observed: a mitochondrial Ca2+ pool and a nonmitochondrial, ATP-dependent, Ins 1,4,5-P3-responsive Ca2+ pool. When the homogenate was separated into microsomes, primary granules, and secondary granules, the nonmitochondrial Ca2+ pumping and the Ins 1,4,5-P3-induced Ca2+ release occurred only in the microsomal fraction. In a gradient developed to separate different microsomal organelles, maximal Ca2+ pumping activity occurred in fractions of low densities. Correlations between Ca2+ uptake and organelle markers were negative for the endoplasmic reticulum (r = -0.49) and positive for plasma membrane (r = 0.47), Golgi (r = 0.62), and endosomes (r = 0.96). Because the Ca2+ pumping organelles in these fractions were insensitive to micromolar vanadate and digitonin treatment, they are unlikely to be plasma membrane vesicles. We conclude first that microsomal fractions of human neutrophils contain organelles that lower the ambient free Ca2+ concentration and respond to Ins 1,4,5-P3. Second, granules are not involved in intracellular Ca2+ regulation in neutrophils. Third, nonendoplasmic reticulum organelles, such as endosomes, Golgi elements, or yet undefined specialized structures, play a major role in intracellular Ca2+ homeostasis in human neutrophils.