Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on sucrose density gradients, and ATP-dependent proton pumping activity was localized by the techniques of [(14)C]methylamine uptake and quinacrine fluorescence quenching. Two peaks of proton pumping activity were detected: a light peak (1.07 grams/cubic centimeter) corresponding to the previously characterized tonoplast-type H(+)-ATPase, and a second peak (1.13 grams/cubic centimeter) which coincided with the Golgi markers, latent UDPase, and glucan synthase I. The second peak was lighter than that of the plasma membrane marker, uridine diphosphoglucose-sterol glucosyltransferase (1.16 grams/cubic centimeter) and was not inhibited by vanadate, an inhibitor of the plasma membrane ATPase. The activity was also better correlated with the Golgi cisternae marker, glucan synthase I, than with latent UDPase, a secretory vesicle marker, but a secretory vesicle location cannot be ruled out. The tonoplast-type and Golgi proton pumps were similar in several respects, including a pH optimum at 7.2, stimulation by chloride, inhibition by diethylstilbestrol and N,N'-dicyclohexylcarbodiimide (DCCD), insensitivity to oligomycin and azide, and nucleotide specificity for Mg(2+)-ATP. However, the Golgi H(+) pump was much less sensitive to nitrate and iodide, and more sensitive to the anion channel blockers, 4-acetamido-4'-isothiocyano-2,2'-stilbene sulfonic acid (SITS) and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) than the tonoplast-type H(+)-pump. The Golgi pump, but not the tonoplast-type pump, was stimulated by valinomycin in the presence of KCl. It is concluded that the Golgi of corn coleoptiles contains a KCl-stimulated H(+)-ATPase which can acidify the interior of Golgi cisternae and associated vesicles.