A beta-mannosidase (beta-D-mannoside mannohydrolase, EC 3.2.1.25) was purified to apparent homogeneity from the culture filtrate of the fungus, Aspergillus niger. The enzyme had an estimated molecular weight of about 120,000 and was a glycoprotein. Radioactive enzyme was prepared by growing the fungus in [14C]fructose, and this enzyme was used for the preparation of 14C-glycopeptides. The glycopeptides were purified on Sephadex G-25 and G-50 and were then hydrolyzed for sugar analysis. Two radioactive sugars were found in the glycopeptides and these were identified as mannose and glucosamine in a ratio of 2.5 or 3:1. Based on susceptibility of the enzyme to alkaline treatment and the formation of [3H]glucosaminitol in the presence of NaB3H4, the oligosaccharide is apparently attached to the protein in a GlcNAc-asparagine linkage. The beta-mannosidase had good activity on p-nitrophenyl-beta-D-mannoside but was inactive on p-nitrophenyl-alpha-D-mannoside as well as on other p-nitrophenyl glycosides. It also showed good activity on the beta(1 leads to 4)-linked trisaccharide of mannose and somewhat lower activity of the corresponding disaccharide. With each of these substrates the Km was about 1 mM, whereas with the p-nitrophenyl-beta-D-mannoside the Km was about 2 mM. The beta-mannosidase also released [14C]mannose from the Man-GlcNAc-GlcNAc trisaccharide isolated from the lipid-linked oligosaccharides of aorta and released mannose from the disaccharides, Man-(beta1 leads to 4)GlcNAc and Man-(beta1 leads to 4)ManNAc. The pH optimum for the enzyme was about 3.5 to 4.0 in glycine or acetate buffer.