Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.