Saccharomyces cerevisiae contains two genes, CITI and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CITI product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikingly at their amino termini. The amino terminus of the CITI primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases. This extension consists of a typical mitochondrial targeting motif. The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E. coli and porcine enzymes. The CIT2-encoded extension is not homologous to that of CITI, resulting in a nonmitochondrial localization of the product. The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences. The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.