The substrate specificities of rat liver microsomal glucosidases I and II, which hydrolyze oligosaccharides of the composition Glc3-1Man9GlcNAc, were investigated. Glucosidases I and II were shown to have a specific and necessary interaction with the 6'-pentamannosyl branch of their oligosaccharide substrates; activity was reduced when mannose residues were removed from this branch. Both glucosidases responded to features at the reducing end of the oligosaccharide substrates; activity was lower towards lipid-linked and peptide-linked substrates than towards free oligosaccharide substrates. In addition, the glucosidases appeared to discriminate between oligosaccharides linked to lipid and oligosaccharides linked to peptide. We conclude that rat liver microsomal glucosidases I and II interact with extensive portions of oligosaccharide structure in addition to the glucose residues which they hydrolyze.