The acidosome, a newly described organelle in Dictyostelium discoideum, is rich in vacuolar proton pumps (V-H(+)-ATPases) and is responsible for the acidification of endocytic vacuoles. Purified acidosomes were not significantly contaminated by lysosomes, endosomes, or plasma membranes but contained a small fraction of contractile vacuole markers. The specific activity of the proton pump in these acidosomes reached 30 mumol/min/mg protein, the highest yet reported for any V-H(+)-ATPase. The V-H(+)-ATPase was the predominant protein in acidosomes. Based on gel electrophoresis and densitometry, its 8 polypeptides had the following apparent molecular mass (in kDa) and stoichiometry: 90(1), 68(3), 53(3), 42(1), 37(3), 25(3), 17(6), and 15(1). These values suggested a Mr congruent to 8 x 10(5), consistent with the hydrodynamic properties and electron microscopic image of the purified pump. The 90- and 17-kDa polypeptides were integral, while the others were peripheral; only the 90-kDa subunit was biosynthetically labeled by [3H]glucosamine and 35SO4. The specific content of phosphatidylcholine and phosphatidylserine in the acidosomes was the highest of any subcellular fraction tested, while sterols and sphingolipids were the lowest. Acidosomes had congruent to 10% of the lipid biosynthetically labeled with [3H]glucosamine. This organelle contributed 5% of cellular protein and 15% of the phospholipid in stationary cultures. We conclude that the acidosome in Dictyostelium is a biochemically discrete organelle, produced by the endoplasmic reticulum/Golgi apparatus but distinct from other endomembranes as well as from the plasma membrane.