Gaucher disease (GD) type 1 is the most prevalent lysosomal storage disease and the most prevalent genetic disease among the Ashkenazi Jews. The defective activity of acid beta-glucosidase is the enzymatic basis of GD and is inherited as an autosomal recessive trait. To investigate the genetic basis of Ashkenazi Jewish GD type 1, a cDNA encoding acid beta-glucosidase was isolated from a cDNA library constructed using splenic poly(A)+RNA from a patient. The cDNA was sequenced to identify mutations, and the presence of a single missense mutation in the patients' genome was confirmed by selective oligonucleotide hybridization and by restriction endonuclease digestion analyses of amplified genomic sequences. This G----A transition (Arg-119 to Gln-119) was present heterozygously in the index patient and his affected third cousin but was not present in normal non-Jewish individuals. This mutation is the second single base mutation found in Ashkenazi Jewish GD type 1 patients. Furthermore, results obtained with the affected third cousin suggest that at least three mutant alleles may be present in this GD subpopulation.