Thiolase from pig heart shows in dilute salt solutions the following hydrodynamic properties: s°20,w= 7.88 S D°20,w= 4.2 × 10−7 cm2/sec = 0.731 ml/g From these data a molecular weight of 169,000 was calculated which is in good agreement with the value of 168,000 obtained by the Archibald method.In the presence of 5 M guanidine‐HCl the enzyme dissociates into subunits with a molecular weight in the range of 39,000–44,000 as determined by use of the Archibald method, sedimentation equilibrium analysis and combination of sedimentation velocity with viscosity and diffusion measurements.The sedimentation coefficient and the molecular weight of the subunits in 5 M urea (s°20,w= 1.86 S, mol. wt. = 43,700) and in 70% formic acid (s°20,w= 1.73 S, mol. wt. = 41,700) are comparable to those obtained in the presence of guanidine‐HCl.Treatment of the enzyme with a large excess of succinic anhydride caused a splitting into protein particles with a molecular weight of approximately 92,000, corresponding to half the size of the native enzyme.Quantitative determinations of the NH2‐terminal amino acid residues by reaction with dinitrofluorobenzene and by Edman degradation yielded 4 valine residues per mole of protein.These results indicate that thiolase from pig heart contains 4 subunits of similar size, each consisting of one polypeptide chain. The subunits are not linked by covalent bonds. Although binding studies using [1‐14C]acetyl‐CoA and iodo‐[1‐14C]acetamide have indicated the presence of at least 3 active sites per mole of enzyme, we propose that the fully active tetramer contains one active site per subunit.The dissociation of thiolase in 5 M urea is a reversible process. After dilution to a urea concentration of 0.1 M the reconstitution of the tetrameric structure can be followed by enzyme assay and sedimentation analysis. The association of the subunits is dependent on protein concentration, temperature, pH and the nature of the buffer. Under optimal conditions the original enzyme activity can be restored completely.Thiolase dissociated with 5 M guanidine‐HCl or 70% formic acid could be reactivated in similar experiments to 70% and 50% of the original activity, respectively.Denaturation by heat or treatment with trichloroacetic acid could also be partially reversed (40%, 70%) after dissolving the precipitated protein in 5 M urea.