3-Glucuronidase from human placenta was purified 18 000-fold to homogeneity in three steps: (1) batch immunoadsorption on antibody-Sepharose resin; (2) DEAE-Sephadex chromatography; and (3) reduction and alkylation followed by DEAE-Sephadex chromatography. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis, (b) gel filtration on Sephadex G-200, (c) sedimentation equilibrium data, and (d) double immunodiffusion against either goat or rat antibody to 3glucuronidase. The molecular weight determination gave values of 310 000 ± 10 000 (gel filtration) and 286 000 ± 10 000 (sedimentation equilibrium). Amino acid analysis showed no cysteine. No radioactivity was incorporated into the protein upon reduction and alkylation with [14C]iodoacetic ^/-Glucuronidase (EC 3.2.1.31) is a lysosomal acid hydrolase which has been studied extensively in different mammalian systems. Rat liver 3-glucuronidase (de Duve et al., 1955) and mouse liver 3-glucuronidase (Paigen, 1961;Fishman et al., 1969) are found in both lysosomes and microsomes. Mouse liver 3-glucuronidase has multiple forms (Swank and Paigen, 1973), tetrameric structure, carbohydrate residues, and only trace amounts of cysteine (Tomino et al., 1975). The 3-glucuronidase from rat liver (Stahl and Touster, 1971;Owens et al., 1975) or rat preputial gland (Himeno et al., 1974;Tulsiani et al., 1975) has chemical, physical, and biochemical properties similar to the mouse enzyme. 3-Glucuronidase from bovine liver (Plapp and Cole, 1966) was found to have multiple forms which differed in carbohydrate content and isoelectric point (Plapp and Cole, 1967). Human placental enzyme has been partially purified (Contractor and Shane, 1972;Contractor and Oakey, 1977) and the human liver enzyme purified to apparent homogeneity (Musa et al., 1965). However, the properties of the latter differ from those we report here, post From the