We report the isolation of a novel human cDNA encoding a type II membrane protein of 79.5 kDa with amino acid sequence similarity to Class I alpha 1,2-mannosidases. The catalytic domain of the enzyme was expressed as a secreted protein in Pichia pastoris. The recombinant enzyme removes a single mannose residue from Man9GlcNAc and [1H]-NMR analysis indicates that the only product is Man8GlcNAc isomer B, the form lacking the middle-arm terminal alpha 1,2-mannose. Calcium is required for enzyme activity and both 1-deoxymannojirimycin and kifunensine inhibit the human alpha 1,2-mannosidase. The properties and specificity of this human alpha 1,2-mannosidase are identical to the endoplasmic reticulum alpha 1,2-mannosidase from Saccharomyces cerevisiae and differ from those of previously cloned Golgi alpha 1,2-mannosidases that remove up to four mannose residues from Man9GlcNAc2 during N-glycan maturation. Northern blot analysis showed that all human tissues examined express variable amounts of a 3 kb transcript. This highly specific alpha 1,2-mannosidase is likely to be involved in glycoprotein quality control since there is increasing evidence that trimming of Man9GlcNAc2 to Man8GlcNAc2 isomer B in yeast cells is important to target misfolded glycoproteins for degradation.