Chicken liver P-protein of the multienzyme glycine cleavage system catalyzes the first partial reaction of glycine cleavage. In the partial reaction, glycine and H-protein serve as substrates and the products are CO2 (not bicarbonate) and the decarboxylated portion of glycine attached to H-protein. The reaction exhibited Michaelis-Menten kinetics with respect to both substrates. The optimum pH for the reaction is 7.1, with 6.5 for the reverse reaction. Km values for glycine and H-protein are independent of the concentration of the the co-substrate, and calculated values are 5.8 mM for glycine and 3.4 microM for H-protein. Initial velocity experiments gave intersecting double reciprocal plots that conform to a sequential mechanism. Product inhibition studies revealed that both products inhibited competitively with respect to the varied substrate. Glycine methyl ester was found to be a competitive inhibitor of glycine and noncompetitive inhibitor of H-protein. H-protein whose lipoic acid prosthetic group and cysteinyl residues were modified with N-ethylmaleimide was a noncompetitive inhibitor of glycine and a competitive inhibitor of H-protein. These results are most consistent with a sequential random Bi Bi mechanism in which no abortive dead end complex is formed. This was supported by an isotope exchange experiment.