The effects of etomoxiryl-CoA on purified carnitine acyltransferases and on carnitine acyl-transferases of rat heart mitochondria and rat liver microsomes were determined. At nanomolar concentrations, the data agreed with that of other investigators who have shown that etomoxiryl-CoA must be binding to a high affinity site with specific inhibition of mitochondrial carnitine palmitoyltransferase (CPTo). Micromolar amounts of etomoxiryl-CoA inhibited both short- and long-chain carnitine acyltransferases. The concentrations of etomoxiryl-CoA required for 50% inhibition of the different carnitine acetyltransferases and microsomal and peroxisomal carnitine octanoyltransferase were in the low micromolar range. Mixed-type and uncompetitive inhibition kinetics were obtained, depending on the source of purified enzyme. When purified rat heart CPT was incubated with etomoxiryl-CoA, it increased the K0.5 and decreased the Hill coefficient for acyl-CoA. Both proteins and phospholipids of mitochondria and microsomes formed covalent adducts of [3H]etomoxir, with the predominant labeling in phospholipids. None of the purified enzymes formed covalent adducts when incubated with [3H]etomoxiryl-CoA, in contrast to intact mitochondria or microsomes. The major 3H-labeled protein for rat heart mitochondria had a molecular weight of 81,000 +/- 4000, and the major proteins from microsomes had a molecular weight of 51,000-57,000. Malonyl-CoA prevented most of the tritum incorporation into the 81,000 Da protein of mitochondria, but it had little effect on incorporation of tritiated etomoxir into the 51,000-57,000 Da proteins of microsomes. When 50 microM etomoxiryl-CoA was added to microsomes and to mitochondria that had been incubated with radioactive etomoxiryl-CoA, much of the radioactive etomoxir disappeared from the major microsomal proteins, but virtually none was displaced from the mitochondrial protein. Thus, at least two different types of covalent etomoxir complexes were formed. This pulse-chase experiment showed that the mitochondrial protein-etomoxir complex was not turned over, consistent with other data showing that etomoxir inhibited carnitine palmitoyltransferase. In contrast, the major protein-etomoxir complex in microsomes was turned over during the pulse-chase experiment.