The bile acid-conjugating enzyme cholyl-coenzyme A:glycine/taurine N-acyltransferase previously purified from bovine liver was subjected to a bisubstrate kinetic analysis. Double reciprocal plots of reaction rates as a function of variable concentrations of aminoa acid and cholyl-CoA yielded the nonintersecting type of plots typical of ping-pong reaction mechanisms. A Tetra Uni ping-pong mechanism was supported by further kinetic analysis including demonstration that the enzyme will catalyze the release of CoA from cholyl-CoA without glycine being present. This half-reaction is shown not to be the result of an acyl-CoA thiolase contaminating the N-acyltransferase preparation. The specificity of the enzyme for amino acid was found to be quite narrow; no detectable rate was found for D-Ala, L-Ala, L-Ser, L-Glu-NH2, L-ornithine, or beta-alanine. Activity toward various acyl-CoA derivatives was also tested. Phenylacetyl-CoA, benzoyl-CoA, and acetyl-CoA were not substrates. CoA derivatives of the other common bile acids were very good substrates. Conjugated bile acids were efficient competitive inhibitors of cholyl-CoA binding. Cholic acid also inhibited; however, its inhibition pattern was complex. The enzyme could be reversibly inhibited by p-mercuribenzoate and both cholyl-CoA and glycine protected. N-Ethylmaleimide and iodoacetate were not inhibitory.