Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD). We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracellular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant.We stably expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD.Endoglycosidase H digestion and immunofluorescence staining revealed the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH(2)Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase I, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man(8)GlcNAc(2) targets the mutant for ERAD, however, the interaction with calnexin/calreticulin through monoglucosylated oligosaccharides may not be required for the degradation of the mutant.