AbstractCitrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such asin vitrostability, tolerance to changes in pH, degree of self‐polymerization, etc., citrate syntheses different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato andEscherichia coliwere investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (α‐PCS) or yeast citrate synthase‐2 (α‐YCS‐2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating fromE. colicompared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate syntheses. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS‐1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS‐2). These findings are discussed in terms of the possible mechanism of evolution o