An endo-alpha 1,2-mannosidase, which is involved in N-linked oligosaccharide processing, has been purified to homogeneity from crude pig liver microsomes using conventional techniques. Two catalytically active polypeptides, of 48 kDa, have been isolated which degrade [14C]Glc3-1-Man9,-GlcNAc2 to [14C]Glc3-1-Man and a specific Man8-GlcNAc2 isomer. They are not, however, active on synthetic alpha-mannosides. [14C]Glc1-Man9-GlcNAc2 was found to be approximately sevenfold more rapidly hydrolyzed than the [14C]Glc2- and [14C]Glc3-homologues. The 48 kDa and 50 kDa proteins are not N-glycosylated and ran on Superdex 75 as monomers. Kinetic studies showed that these proteins had similar catalytic properties: (i) the pH optima were found to be close to 6.5; (ii) neither activity was metal ion dependent; (iii) hydrolysis of [14C]Glc3-Man9-GlcNAc2 was inhibited strongly by Glc-alpha 1,3-Man (app. Ki approximately 120 microM), but not by 1-deoxymannojirimycin or swainsonine. Other evidence, including immunological data, strongly suggests that the 48 kDa and 50 kDa polypeptides are proteolytic degradation products of a single endo-alpha 1,2-mannosidase, rather than distinct subunits of an oligomeric complex. Possible functions of the endo-alpha 1,2-mannosidase in N-linked oligosaccharide processing are discussed.