The amino acid conjugation of the phenoxyherbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) by animals was examined at the level of the enzymes catalyzing the reactions. The phenoxyherbicides were not substrates for the bile acid conjugating system but were substrates for the mitochondrial xenobiotic conjugating system. The two mitochondrial xenobiotic-CoA: amino acid N-acyltransferases (benzoyltransferase and phenylacetyltransferase) were separated and tested for activity towards 2,4-D-CoA and 2,4,5-T-CoA. The phenylacetyltransferase showed activity towards phenylacetyl-CoA, phenoxyacetyl-CoA and 2,4-D-CoA, but not 2,4,5-T-CoA. Benzoyltransferase conjugated both 2,4-D-CoA and 2,4,5-T-CoA. The overall rates of conjugation of the phenoxyherbicides were slow relative to the standard substrates with both enzymes. This slow rate was found to be due in both cases to a relatively high Km for glycine, and a very slow catalytic rate constant. Both enzymes did, however, have a very high affinity for 2,4-D-CoA and 2,4,5-T-CoA so these compounds proved to be potent alternate substrate inhibitors of both enzymes. The data show that the inefficient in vivo conjugation of the phenoxyherbicides relative to structurally similar compounds can be understood in terms of the kinetic properties of the mitochondrial N-acyltransferases. Further, the potential for the interference of the phenoxyherbicides with the conjugation of other compounds is revealed.