Insulin secretion and B-cell calcium distribution, assessed with the pyroantimonate precipitation technique, were studied in rat pancreases perfused with glucose (3.3 or 16.6 mM) alone or together with verapamil or trifluoperazine (TFP). Total calcium pyroantimonate precipitates (CPP), and those bound to every B cell structure, at every sampling period, were larger at 16.6 mM glucose concentration. The largest percentage of CPP was located, at early stages of the glucose stimulatory period, mainly within the clear halo of the B granules, while later on they shifted to the plasma membrane. Verapamil and TFP diminished the second phase of glucose-induced insulin release and greatly affected the above mentioned pattern of B cell CPP distribution. The main changes consist in a diminution in the total number of CPP all throughout the perfusion-time as well as an alteration in the percentage distribution of the CPP within the different B cell organelles, i.e., an early diminution in the CPP present in the B granules and of those attached to the plasma membrane and to the mitochondria at the end of the perfusion, were the most striking changes observed. The results suggest that during the glucose stimulus, different B cell structures take over the control of available calcium within the cell, following a chronological sequence. Such sequence might be determined by the different Ca2(+)-set point of those structures. Intracellular provision of calcium might be sufficient to maintain the early phase of insulin secretion when the cation entrance is blocked. Conversely, this substitution might not be enough to sustain the second phase of insulin release. The different amounts of CPP in every B cell organelle, besides their buffering capacity to control free Ca2+ availability, might also be coupled to a regulatory role of the cation upon their respective metabolic functions. In some cases, this latter effect may be the main role for the cation distribution. Our results support the concept that the level of free calcium, acting as the coupler for the stimulussecretion process, might be regulated by different calcium pools located in the B cell.