Saccharomyces cerevisiae produces two L-asparaginases (ASPs), intracellular ASP I and cell-wall ASP II. In this report, the ASP-I-encoding gene, ASP1, has been identified by homology cloning based on the structures of ASPs from other organisms. Its deduced protein product has a subunit M(r) of 41,414, and shows substantial sequence homology to the bacterial amidohydrolase family. The product of the S. cerevisiae ASP3 gene, a further member of this family, encoding the nitrogen catabolite-regulated cell-wall ASP II, has 46% overall sequence identity to ASP1. Duplication of ancestral asparaginase genes, resulting in separate intra- and extracellular isozymes, appears to have occurred independently in the prokaryotic and eukaryotic lineages. Exact physical mapping of the new cloned ASP1 gene locates it 73% of the distance from the left telomere of chromosome IV, at a position precisely matching the known genetic map location of ASP1. This, along with the structural features of the clone, confirms that ASP1 is the structural gene encoding cytoplasmic ASP I in S. cerevisiae. Sequence analysis of the ethylmethanesulfonate-induced asp1-12 allele of strain XE101-1A revealed a C-->T transition altering Ala176 to Val. This residue lies within a highly conserved region, and the results suggests a critical function for Ala176 in ASP function. Expression of ASP1 and other recombinant ASPs may allow access to improved products for use in the chemotherapy of leukaemia.