Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.