As part of our ongoing efforts to characterize the N-glycosylation pathway of lepidopteran insect cells, we have isolated an alpha 1,2-mannosidase homolog from an Sf9 cDNA library. This cDNA contains an open reading frame which encodes a 670 amino acid protein with a calculated molecular weight of 75,225 Da. This protein has two potential N-glycosylation sites, two consensus calcium binding sequences, and is predicted to be a type II integral membrane protein with a 22 amino acid transmembrane domain (residues 31-52). The amino acid sequence of this protein is 35-57% identical to Drosophila, human, murine, and yeast alpha 1,2-mannosidases. A transcript of approximately 6 kilobases was detected by Northern blot analysis of Sf9 mRNA. Genomic Southern blots probed with an intron-free fragment of the alpha 1,2-mannosidase gene indicated that there are at least two copies or cross-hybridizing variants of this gene in the Sf9 genome. In vivo expression of the cDNA using a recombinant baculovirus produced a protein that released [3H]mannose from [3H]Man9GlcNAc. This activity required calcium, but not magnesium, and was inhibited by 1-deoxymannojirimycin. These results indicate that Sf9 cells encode and express an alpha 1,2-mannosidase with properties similar to those of other eukaryotic processing alpha 1,2-mannosidases.