In previous work (Herscovics et al., J. Biol. Chem., 269, 9864-9871), a novel mouse alpha-mannosidase cDNA was isolated by homology, taking advantage of identical regions between the amino acid sequences of the yeast and rabbit liver processing alpha 1,2-mannosidases of different specificities to design degenerate oligonucleotides for reverse transcription/polymerase chain reaction. The cDNA isolated from a mouse 3T3 cDNA library encodes a 73 kDa type II membrane protein with a cytoplasmic region of approximately 35 amino acids and a large C-terminal region that contains a consensus Ca(2+)-binding sequence. To study the properties of this enzyme, the C-terminal part lacking the transmembrane region (beginning at either amino acid 106 or 171) was transiently expressed in COS cells as a secreted protein A fusion protein, and the enzymatic properties of the fusion protein bound to IgG-Sepharose were investigated. The enzyme is an alpha 1,2-mannosidase that trims Man9GlcNAc to Man5 GlcNAc (where Man is mannose and GlcNAc is N-acetyl glucosamine). The activity requires divalent cations since it is greatly inhibited by ethylene diamine tetraacetic acid (EDTA). Although Ca2+ is the most effective, the enzyme may also use Mg2+, Mn2+ or Co2+, but not Zn2+, which is inhibitory. The enzyme is inhibited by 1-deoxymannojirimycin, but not by swainsonine. We propose that this novel alpha 1,2-mannosidase cDNA encodes mouse Golgi alpha-mannosidase IB.