The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding partner binder of ARL2 (BART) are ubiquitously expressed in rodent and human tissues and are most abundant in brain. Both ARL2 and BART are predominantly cytosolic, but a pool of each was found associated with mitochondria in a protease-resistant form. ARL2 was found to lack covalent N-myristoylation, present on all other members of the ARF family, thereby preserving the N-terminal amphipathic alpha-helix as a potential mitochondrial import sequence. An overlay assay was developed to identify binding partners for the BART.ARL2.GTP complex and revealed a specific interaction with a protein in bovine brain mitochondria. Purification and partial microsequencing identified the protein as an adenine nucleotide transporter (ANT). The overlay assay was performed on mitochondria isolated from five different tissues from either wild-type or transgenic mice deleted for ANT1. Results confirmed that ANT1 is the predominant binding partner for the BART.ARL2.GTP complex and that the structurally homologous ANT2 protein does not bind the complex. Cardiac and skeletal muscle mitochondria from ant1(-)/ant1(-) mice had increased levels of ARL2, relative to that seen in mitochondria from wild-type animals. We conclude that the amount of ARL2 in mitochondria is subject to regulation via an ANT1-sensitive pathway in muscle tissues.