Two benign mutations, C739T(R247W) and C745T-(R249W), in the ␣-subunit of -hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of G M2 gangliosidosis, as Hex S (␣␣) and Hex A (␣) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo--N-acetylglucosaminide (4-MUGS) by 60 -80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the ␣-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl--N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo--N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the ␣-subunit protein by affecting its stability in vivo, not by affecting the processing of the ␣-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.