The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER). Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991). To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of [35S]methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product. It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes. This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent. These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.