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Thiolase was purified 600–700 fold from a crude extract of pig heart by use of differential adsorption on Al(OH)3 gel, ammonium sulfate fractionation and ion exchange chromatography on DEAE­Sephadex and phosphorylated cellulose. The enzyme preparation is homogenous by the following criteria: sedimentation analysis and electrophoresis on polyacrylamide gel and on cellulose acetate membranes. The isoelectric poit of the protein was found to be approximately pH 7.2–7.3. The enzyme crystallized from ammonium sulfate solutions as thin plates of irregular shape. Crystalline thiolase stored for 18 months at –10° did not show any loss of activity. The thiolytic activity and the acyl transferase activity of the enzyme could be demonstrated separately. The thiolytic activity was demontrated by the specific incorporation of 14C from [1 ­14C]­acetyl­CoA into the carboxyl position of unlabelled acetoacetyl­CoA on incubation wtih the enzyme. The acyl transferase activity eas studied using either [1­14C]­CoA and patethein as substrates or 14C­labelled CoA and acyl­CoA derivatives of varius chain length. A remarkable specificity was found for the transfer of the acetyl group. Thiolase activity and acyl transferase activity of the enzyme were diminished with the same kinetics during heat treatment or incubation with N ­ethylmaleeimide, ρ­cholomercuribenzoate or iodoacetamide. this indicates that both activities belong to the same protein. Inhibition of thiolase and acyltranferase activities by iodoacetamide (5 x 10­5 could be prevented by preicubation of the enzyme with acetoacetyl­CoA or acetyl­CoA. The acyl transfer is the rate determining step when the thiolase reaction operates in the direction of chain cleavage. Incubation of thiolase with acetyl­Coa results in binding of acetyl groups to the protein. The uptake of acetyl groups is dependent upon the specific activity of the enzyme and corresponds to 3 acetyl residues per molecule of protein (molecular weight 170,000) for the highest enzymatic activity obtained. both incorporation of acetyl groups into the enzyme and thiolase activity are inhibited to the same extent by iodoacetamide. the incorportion of radioactivity into the enztme during incubation with iodo­[1­14C]­acetamide can be divided into two different reactions: a fast incorporation with accompanying loss of enzymatic and a much slower process which continues after complete inactivation. From the initial rate of incorporation, it was calculaetd that incorporation of 3.1 carbamoylmethyl residues per molecule of protein would completely inhibit the enzyme withthe highest obtained activity. this ration cmpares well with the results obtained by acetyl binding studies and indicates a minimum of 3 active sites per molecule of enzyme. Enzyme labelled with iodo­[1­14C]acetamide was hydrolyzed with 6 N HCl. Approximately 95% of the radioactivity in the hydrolysate was found to coincide with S­carboxymethyl­L­cystine on electrophoresis and paper chromatography. Repeated crystallization with authentic carboxymethyl­cystine did not result in a decrease of specific radioactivity. [ABSTRACT FROM AUTHOR]