A combination of equilibrium ultracentrifugation and polyacrylamide gel electrophoresis techniques has been used to establish the quaternary structure of citrate synthase from acetate-grown Escherichia coli K12 3000. In polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), the pure enzyme showed one major band whose mobility was consistent with a molecular weight of 46,000 plus or minus 2000 g/mol, and a little material of 87,000 plus or minus 5000 g/mol. When first cross-linked with dimethyl suberimidate and then submitted to electrophoresis in SDS, citrate synthase showed six bands, in widely different amounts, whose apparent molecular weights were almost integral multiples of 47,000 g/mol. The dimer was the major product of the cross-linking procedure. In 6 M guanidine HCl at pH 7.0, citrate synthase behaved as a single component in high-speed sedimentation equilibrium experiments, with a weight average molecular weight of 43,400 plus or minus 300 g/mol. The molecular weight of native citrate synthase was investigated by high-speed sedimentation equilibrium ultracentrifugation under different conditions of pH and KCl concentration. In 0.02 M Tris-Cl at pH 7.0 and 7.8, the enzyme was a mixture of oligomers, with species ranging from monomer (47,000 g/mol) to greater than decamer being present. At pH 9.0, only dimer was seen (94,000 g/mol). Large aggregates were present at pH 10.0. The addition of small amounts of KCl, a potent activator of the enzyme, simplified the mixture of oligomers considerably at pH 7.8. A detailed analysis of the data with 0.05 M KCl indicated that dimer and hexamer were the only species present, with marked nonideality. Increasing the KCl concentration to 0.10 M converted all the enzyme to hexamer. The amino acid composition of E. coli citrate synthase was presented. Taken together with peptide mapping experiments of others (J. A. Wright and B. D. Sanwal (1971), J. Biol. Chem. 246 1689), it indicates that the subunits have all the same or very similar amino acid sequences. The dansylation method revealed only methionine at the N-termini of the citrate synthase polypeptide chains. Citrate synthase from E. coli thus resembles the enzyme from eukaryotes in that it consists of subunits weighing just under 50,000 g/mol, although these subunits are more highly aggregated in the bacterial enzyme under most conditions. This conclusion is in disagreement with that of Wright and Sanwal (1971, see above), who reported a subunit size of 62,000 g/mol.