Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp6G' docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp6tO' NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60" binding protein. An analysis of pp6OV' binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp6@' NH2 terminus suggests alternative strategies for uncovering pp6G0 membrane binding species.Cellular transformation by the Rous sarcoma virus (RSV) is mediated by the membrane-bound tyrosine kinase pp6ov-srC, a