To determine the role of N-linked oligosaccharides in the folding of glycoproteins, we analyzed the processing of in vitro translated influenza hemagglutinin (HA) in dog pancreas microsomes. We found that binding to calnexin, a membrane-bound molecular chaperone, was specific to molecules that possessed monoglucosylated core glycans. In the microsomes, these were generated either by glucose removal from the original triglucosylated core oligosaccharide by glucosidases I and II or by reglucosylation of already unglucosylated high mannose glycans. Release of fully folded HA from calnexin required the removal of the remaining glucose by glucosidase II. The results provided an explanation for trimming and reglucosylation activities in the endoplasmic reticulum and established a direct correlation between glycosylation and folding.