Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyses the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. A cloned complementary DNA sequence encoding Drosophila citrate synthase has been isolated from a Drosophila cDNA expression library in lambda ZAPII. The nucleotide sequence of the 1.14-kilobase cDNA including the active site of the enzyme was determined. Comparison of the deduced amino acid sequence of Drosophila citrate synthase cDNA with those of the yeast and pig heart enzymes revealed extensive homology, 72% and 59% with pig heart and with yeast enzymes, respectively. In contrast, only 20% of the Drosophila enzyme residues were identical to the functionally similar E. coli enzyme residues. The active site residues, however, exhibited strict conservation during evolution.