Long chain fatty acids (LCFAs) are an important energy substrate used by cardiac myocytes and other cells, but the mechanism whereby these molecules cross the plasma membrane is poorly understood. We used an expression cloning strategy and a cDNA library from 3T3-L1 adipocytes to identify a cDNA that, when expressed in cultured cells, augments uptake of LCFAs. This cDNA encodes a novel 646 amino acid fatty acid transport protein (FATP) with six predicted membrane-spanning regions and that is integrally associated with membranes. Immunocytochemistry and subcellular fractionation of 3T3-L1 adipocytes show that FATP is localized to the plasma membrane. We propose that FATP is a plasma membrane transporter for LCFAs.