The Saccharomyces cerevisiae processing αl,2-mannosidase, which trims Man GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmem-brane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the a-factor promoter and signal peptide in a high-copy vector for expression in S.cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in α-mannosidase activity that was directly related to the increase in cell density, whereas no α-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized α-mannosidase. The recombinant α-mannosidase was present in the medium at a level of 1 mg/1 and was purified in a single step by chromato-graphy on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific αl,2-mannose residue of the al,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by 5 kDa, showing that the heterogeneity is not due to differential W-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations. The Km for the Man9GlcNAc substrate was 0.3 mM. These results demonstrate that the recombinant αl,2-mannosidase has the same properties as the endogenous processing enzyme.