Mutations of the gene encoding the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase, are the cause of Tay-Sachs disease. We previously showed that fibroblasts from one patient (WG1051) synthesized an unstable alpha-subunit that was smaller than normal and appeared to be trapped in an early biosynthetic compartment (Zokaeem, G., Bayleran, J., Kaplan, P., Hechtman, P., and Neufeld, E. F. (1987) Am. J. Hum. Genet. 40, 537-547). We now have identified the mutation as a deletion of cytosine at position 1510 of the coding sequence. We first determined that the structural abnormality was at the carboxyl terminus of the protein and then sequenced the corresponding regions of the cDNA and genomic DNA after amplification by the polymerase chain reaction. The frameshift mutation, which is present on both alleles, causes premature termination four codons downstream, and the loss of a very hydrophilic stretch of 22 amino acids. Expression of alpha-subunit cDNA with the cytosine deletion in Cos-1 cells reproduced the WG1051 phenotype, i.e. a truncated alpha-subunit that was retained and degraded in an early compartment, presumably the endoplasmic reticulum. Loss of the cysteine residue at position 522 was not the sole cause of instability and defective transport.