Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.