A cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 85,000 by gel filtration on Sephadex G-150 and, after treatment with 2-mercaptoethanol, about 38,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. These results indicate that the native enzyme is composed of two apparently identical monomeric units. The protein band could also be stained with the Schiff reagent after periodate oxidation, indicating that the proteinase is a glycoprotein. Analyses showed that the enzyme contains approximately 4 glucosamine and 16 mannose residues per molecule (M.W. 85,000). The amino acid composition generally resembled those of cathepsins D except for the lower contents of basic amino acid residues, especially lysine. It also showed some similarity to pepsinogens. The optimal pH was 2.3--3.5 with hemoglobin as a substrate. The enzyme was strongly inhibited, like pepsin, by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as well as by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. The proteinase was stable in alkaline solution up to pH 9.0, and was rather stable to sequential acidification and neutralization. The acidification resulted in an increase of electrophoretic mobility towards the anode.