Cathepsin H was isolated from human placenta by autolysis, acetone fractionation, and chromatography on DEAE-cellulose, Sephadex G-75, hydroxyapatite and concanavalin A-Sepharose. The enzyme gave on SDS-polyacrylamide gel electrophoresis two bands of Mr 25,500 and 28,500. Two active forms of the enzyme, with pI of 6.0 and 6.45, were obtained by isoelectric focusing. The enzyme is stable over the pH range 5-7.5, whereas it becomes inactive on heating to 50 degrees C. Cathepsin H of human placenta, like the enzyme from other sources, hydrolyses protein and naphthylamide substrates, showing within the latter group the strongest preference towards arginine-beta-naphthylamide (pH optimum 6.8). The enzyme is inhibited by the known inhibitors of cysteine proteases and by placental cystatins.