Chronic and adult-onset GM2 gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of beta-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective alpha-subunit that failed to associate with the beta-subunit. We have now found a guanosine to adenosine transition at the 3' end of exon 7, which causes substitution of serine for glycine at position 269 of the alpha-subunit [designated 269 (Gly----Ser) substitution]. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly----Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly----Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic GM2 gangliosidosis. It was not found in beta-hexosaminidase A-deficient cells obtained from patients with infantile Tay-Sachs disease nor in cells from individuals who do not have beta-hexosaminidase A deficiency. However, there must be additional mutations with similar consequences, since the 269 (Gly----Ser) substitution was not present in fibroblasts from two patients with juvenile GM2 gangliosidosis even though these had an association-defective alpha-subunit.