The GM2 gangliosidoses are caused by mutations in the genes encoding the alpha- (Tay-Sachs) or beta- (Sandhoff) subunits of heterodimeric beta-hexosaminidase A (Hex A), or the GM2 activator protein (AB variant), a substrate-specific co-factor for Hex A. Although the active site associated with the hydrolysis of GM2 ganglioside, as well as part of the binding site for the ganglioside-activator complex, is associated with the alpha-subunit, elements of the beta-subunit are also involved. Missense mutations in these genes normally result in the mutant protein being retained in the endoplasmic reticulum and degraded. The mutations associated with the B1-variant of Tay-Sachs are rare exceptions that directly affect residues in the alpha-active site. We have previously reported two sisters with chronic Sandhoff disease who were heterozygous for the common HEXB deletion allele. Cells from these patients had higher than expected levels of mature beta-protein and residual Hex A activity, approximately 20%. We now identify these patients' second mutant allele as a C1510T transition encoding a beta-Pro504 --> Ser substitution. Biochemical characterization of Hex A from both patient cells and cotransfected CHO cells demonstrated that this substitution (a) decreases the level of heterodimer transport out of the endoplasmic reticulum by approximately 45%, (b) lowers its heat stability, (c) does not affect its Km for neutral or charged artificial substrates, and (d) lowers the ratio of units of ganglioside/units of artificial substrate hydrolyzed by a factor of 3. We concluded that the beta-Pro504 --> Ser mutation directly affects the ability of Hex A to hydrolyze its natural substrate but not its artificial substrates. The effect of the mutation on ganglioside hydrolysis, combined with its effect on intracellular transport, produces chronic Sandhoff disease.