T-protein, a component of the glycine cleavage system, has been purified to apparent homogeneity from chicken liver mitochondria. The molecular weight was 37,000 by sedimentation equilibrium centrifugation and 38,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a molecular weight of 41,000, indicating that the protein is composed of a single polypeptide. A partial specific volume of 0.73 ml/g was obtained from the amino acid composition. The absorbance coefficient, A1%280nm, is 6.63 in 50 mM potassium phosphate buffer, pH 7.0. T-protein is a basic protein having a pI value of 9.8 and relatively rich in arginine rather than lysine. The protein catalyzed the degradation of the protein-bound intermediate (-CH2NH2 moiety of glycine) to a 1-carbon unit and NH3. The reaction is dependent on tetrahydrofolate (H4-folate). Apparent Km values for the intermediate and H4-folate were 3.7 microM and 0.17 mM, respectively. T-protein associated with H-protein forming a complex which was composed of one molecule each of T-protein and H-protein. Formation of the complex was not affected by the modification of the cysteinyl residues on H-protein with N-ethylmaleimide.