Juvenile GM2 gangliosidosis is a rare neurodegenerative disorder closely related to Tay-Sachs disease but of later onset and more protracted course. The biochemical defect lies in the alpha-subunit of the lysosomal enzyme beta-hexosaminidase. Cultured fibroblasts derived from patient A synthesized an alpha-subunit which could acquire mannose 6-phosphate and be secreted, but which failed to associate with the beta-subunit to form the enzymatically active heterodimer. By contrast, fibroblasts from patient B synthesized an alpha-subunit that was retained in the endoplasmic reticulum. To identify the molecular basis of the disorder, RNA from fibroblasts of these two patients was reverse-transcribed, and the cDNA encoding the alpha-subunit of beta-hexosaminidase was amplified by the polymerase chain reaction (PCR) in four overlapping fragments. The PCR fragments were subcloned and shown by sequence analysis to contain a G to A transition corresponding to substitution of histidine for arginine at position 504 in the case of patient A and at position 499 in the case of patient B. The mutations were confirmed by hybridization of allele-specific oligonucleotides to PCR-amplified fragments of DNA corresponding to exon 13 of the alpha-subunit gene. The Arg504----His mutation was found on both alleles of patient A as well as of another unrelated patient; the homozygosity of this mutant allele is attributable to consanguinity in the two families. The Arg499----His mutation was found in patient B in compound heterozygosity with a common infantile Tay-Sachs allele. There is additional heterogeneity in juvenile GM2 gangliosidosis, as neither mutation was found in the DNA of a fourth patient. The Arg----His mutations at positions 499 and 504 are located at CpG dinucleotides, which are known to be mutagenic "hot spots."