Mitochondrial citrate synthase (EC 4.1.3.7) represents the first enzyme of the tricarboxylic acid cycle, catalyzing the condensation of acetyl-CoA and oxaloacetate, finally yielding citrate and CoA. We report here the isolation of cDNA clones encoding citrate synthase from Nicotiana tabacum, Beta vulgaris and Populus. Nucleotide and deduced amino acid sequences were compared with previously published sequences of mitochondrial citrate synthases from Arabidopsis thaliana and potato, as well as with the sequence of glyoxysomal citrate synthase from pumpkin. Homologies between the various plant mitochondrial enzymes were in the range from 77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco) on the nucleotide level (coding regions only), and in the range from 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs. tobacco, respectively) on the amino acid level. Identities of the mitochondrial isozymes to the pumpkin glyoxysomal enzyme were below 30% on the nucleotide and amino acid level. In Northern blot experiments citrate synthase mRNA was detected in all tissues analyzed. However, levels of expression showed tissue dependency despite the fact that citrate synthase is usually considered a house-keeping enzyme. Whether these different levels of expression reflect tissue-specific variations with respect to basic metabolism awaits further analysis.