Abstract: Disclosed is the preparation of eukaryotic cells which are capable of producing smaller and less heterogenic N-linked glycans, particularly glycans having the structure Man.sub.3 GlcNAc.sub.2. In particular embodiments, CHO cells are produced having two particular defects in enzymes involved in N-linked glycosylation. The first defect, PIR, leads to an accumulation of Man.sub.5 GlcNAc.sub.2 -P-P-dolichol and thus lends resistance to glycosylation processing inhibitors such as castanospermine and swainsonine. The second defect involves a defect in the enzyme N-acetylglucosaminyltransferase I activity, and thus prevents the attachment of N-acetylglucosamine residues to oligosaccharide structures. About 70 to 75% of the N-linked glycans produced by cells of the present invention are thus in the form of Man.sub.3 GlcNAc.sub.2, making these lines particularly well suited to the production of proteins, e.g.