CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.Long chain fatty acids, an important energy source in mitochondria, are oxidized in the matrix via -oxidation. Long chain fatty acids are activated on the cytosolic side of the mitochondrial outer membrane (MOM) 2 by long chain acyl-CoA synthetase (ACSL) (1). To be metabolized, activated fatty acids, as well as other substrates, ions, and nucleotides, cross the MOM through the voltage-dependent anion channel (VDAC), also called mitochondrial porin.Historically, the MOM was considered to be a sieve through which small compounds passed unimpeded. That view is no longer tenable. In Trypanosoma brucei, which expresses only one VDAC, knock-out of VDAC leads to mitochondria that no longer oxidize substrates (2). However, with disruption of the outer membrane, the mitoplasts are fully capable of oxidizing substrates. These studies highlight the essential role of VDAC in mitochondrial substrate oxidation in transportation of small anion substrates through the MOM.We studied PA22, a 22-kDa polyanion VDAC inhibitor (3), in mitochondrial substrate oxidation and observed that ADPstimulated glutamate, succinate, (ϩrotenone), and palmitoylcarnitine (ϩmalate) oxidation rates were unaffected. However, oxidation of palmitate (ϩATP, Mg 2ϩ , CoA, carnitine, and malate) and palmitoyl-CoA (ϩcarnitine and malate) was inhibited at 1-2 nmol of PA22/mg of mitochondrial protein. These data indicate that PA22 is selective for the first two steps in fatty acid oxidation without affecting other substrates. Additionally, we showed that PA22 interacts with VDAC by demonstrating that binding of hexokinase to rat liver mitochondria was inhibited by PA22 (3). VDAC was initially identified as the hexokinase-binding protein (4).PA10, a 10-kDa polyanion VDAC inhibitor (5), decreases ADP-stimulated oxidation of glutamate, malate, and succinat...