The lysosomal enzyme, beta-hexosaminidase, is composed of two chains, alpha and beta. In Tay-Sachs disease, mutations in the gene encoding the alpha-chain produce a beta-hexosaminidase deficiency that results in the storage of its natural substrate, GM2 ganglioside. To obtain the background information for the eventual identification of the mutational errors in Tay-Sachs disease and to determine possible relationships between protein and gene structure, we have characterized the intron-exon organization of the human beta-hexosaminidase alpha-chain gene. Several overlapping clones were isolated from human genomic libraries constructed in cosmid and bacteriophage vectors. The cloned genomic DNA was analyzed by restriction endonuclease mapping, Southern blotting, and DNA sequencing. It was determined that the alpha-chain gene is approximately 35 kilobases long and is split into 14 exons. Sequences which resemble the "TATA" and "CAAT" transcriptional regulatory motifs are present at the 5' end of the gene. Differential transcription or processing of the most 3' exon of the gene results in two alpha-chain mRNAs with different 3'-untranslated regions. The first exon of the gene encodes the amino-terminal portion of the alpha-chain which is removed during the proteolytic maturation of the enzyme, raising the possibility that this segment may exist as a functional domain.