We have identified a mannosidase in rat liver that releases alpha-1 --> 2, alpha-1 --> 3 and alpha-1 --> 6 linked manose residues from oligosaccharide substrates, Man(n)GlcNAc where n = 4-9. The end product of the reaction is Man-alpha-1 --> 3[Man-alpha-1 --> 6]Man-beta-1 --> 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a K(m) of 65-mu-M and 110-mu-M for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the alpha-1 --> 2 linked residue followed by hydrolysis of alpha-1 --> 3 and alpha-1 --> 6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-alpha-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.