We report on the isolation of a gene encoding yeast malate synthase. A yeast genomic library was screened using a probe homologous to the yeast enzyme obtained by the polymerase chain reaction. The nucleotide sequence of the cloned gene was determined. Computer analysis showed that the isolated gene is identical to the one previously described as DAL7, which is involved in allantoin metabolism [Mol. Cel. Biol. 9 (1989) 3231-3243]. Enzymatic activities of multicopy transformants, Southern analysis and disruption mutants predict the existence of two genes encoding malate synthases that are differentially regulated at the transcriptional level.