The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.