Respiratory defective pet mutants of Saccharomyces cerevisiae assigned to complementation group G5 are deficient in fumarase. A representative mutant from this complementation group was used to clone a nuclear gene (FUM1) whose sequence encodes a protein homologous to bacterial fumarase. Based on the primary structure homology and the elevated levels of fumarase in transformants harboring FUM1 on a multicopy plasmid, this gene is concluded to code for yeast fumarase. In wild type yeast, fumarase is detected in both mitochondria and the soluble postribosomal protein fraction. Several lines of evidence indicate that the two compartmentally distinct fumarases are isoenzyme products of FUM1. Mutations in FUM1 simultaneously abolish both activities. Transformation of a fumarase mutant with a plasmid containing FUM1 leads to increased fumarase activity in mitochondria and in the postribosomal supernatant fraction. Transformation of the same mutant with a plasmid construct in which the region of FUM1 coding for the amino-terminal 17 amino acids of fumarase is deleted results in a preferential increase of nonmitochondrial fumarase. Northern and S1 nuclease analysis of fumarase transcripts in wild type yeast and in a mutant transformed with FUM1 on an episomal plasmid indicate that the gene is transcribed from multiple start sites, some of which are located inside the coding sequence. The major transcript presumed to code for mitochondrial fumarase has a 5'-untranslated leader of 185 nucleotides. The most abundant shorter transcripts have 5' termini from 57 to 68 nucleotides downstream of the first ATG; their translation products lacking the amino-terminal mitochondrial import signal are proposed to target fumarase to the cytoplasm.