endoplasmic reticulum glucosidase ii is composed of a catalytic subunit conserved from yeast to mammals and a tightly bound noncatalytic hdelcontaining subunit trimming of glucoses from nlinked core glycans on newly synthesized glycoproteins occurs sequentially through the action of glucosidases i and ii in the endoplasmic reticulum er we isolated enzymatically active glucosidase ii from rat liver and found that in contrast with previous reports it contains two subunits alpha and beta sequence analysis of peptides derived from them allowed us to identify their corresponding human cdna sequences the sequence of the alpha subunit predicted a soluble protein 104 kda devoid of known signals for residence in the er it showed homology with several other glucosidases but not with glucosidase i among the homologues we identified a saccharomyces cerevisiae gene which we showed by gene disruption experiments to be the functional catalytic subunit of glucosidase ii the disrupted yeast strains had no detectable growth defect the sequence of the beta subunit 58 kda showed no sequence homology with other known proteins it encoded a soluble protein rich in glutamic and aspartic acid with a putative er retention signal hdel at the c terminus this suggested that the beta subunit is responsible for the er localization of the enzyme