Insulin secretion and the pattern of calcium distribution in B cells, assessed with the pyroantimonate precipitation technique, were simultaneously studied in rat pancreases perfused with 3.3 and 16.6 mM glucose solutions of pH 7.4 and 7.8. We have previously demonstrated the blocking effect of the latter pH upon glucose-induced insulin secretion. Glucose (16.6 mM) caused an increase in the total number of calcium pyroantimonate precipitates (CPP), as well as their number bound to different B cell structures, at every sampling period studied, with respect to the 3.3 mM glucose experiments. Extracellular alkalosis strongly inhibited both phases of the B cell response to the glucose stimulus, and greatly affected the distribution of CPP in the cells with respect to the pH 7.4 ones. During the first phase of glucose induced-insulin secretion, most of the CPP appeared within B granules at pH 7.4, while on the development of the second phase of secretion, they appeared mainly attached to the cell plasma membranes. Conversely, in pH 7.8 experiments, at the first minutes of the glucose challenge, CPP appeared principally located in the cytoplasm, being almost absent from the plasma membrane during the second phase of insulin secretion. These observations suggest that during the glucose stimulus, the cell calcium distribution within the B cells followed a clear chronological sequence. Such sequence might be determined, at least in part, according to the different Ca2(+)-set points of the different B cell structures. In our case, the extracellular alkalosis might interfere with the normal intracellular calcium fluxes, which in consequence might impair release of insulin by affecting several B cell functions.