The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)-β subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-β genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of pβ1 into pβ2, the rate-determining step in β folding, was 7 min for wildtype β but 33 min for β lacking both N-linked glycans. The pβ1 → pβ2 half-time was 7.5 min in CHO cells expressing the β subunit missing the Asn13-linked glycan and 10 min for the β subunit missing the Asn30-linked glycan. The inefficient folding of hCG-β lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG-β-folding pathway. Unglycosylated hCG-β was slowly secreted from CHO cells, and β subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG-β core fragment-like protein. However, coexpression of the hCG-α gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG-β lacking N-linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG-β and may be involved in the folding of this β form. These data indicate that N-linked oligosaccharides assist hCG-β subunit folding by facilitating disulfide bond formation.