We have characterized the role of YPR128cp, the orthologue of human PMP34, in fatty acid metabolism and peroxisomal proliferation in Saccharomyces cerevisiae. YPR128cp belongs to the mitochondrial carrier family (MCF) of solute transporters and is localized in the peroxisomal membrane. Disruption of the YPR128c gene results in impaired growth of the yeast with the medium-chain fatty acid (MCFA) laurate as a single carbon source, whereas normal growth was observed with the long-chain fatty acid (LCFA) oleate. MCFA but not LCFA -oxidation activity was markedly reduced in intact ypr128c⌬ mutant cells compared to intact wild-type cells, but comparable activities were found in the corresponding lysates. These results imply that a transport step specific for MCFA -oxidation is impaired in ypr128c⌬ cells. Since MCFA -oxidation in peroxisomes requires both ATP and CoASH for activation of the MCFAs into their corresponding coenzyme A esters, we studied whether YPR128cp is an ATP carrier. For this purpose we have used firefly luciferase targeted to peroxisomes to measure ATP consumption inside peroxisomes. We show that peroxisomal luciferase activity was strongly reduced in intact ypr128c⌬ mutant cells compared to wild-type cells but comparable in lysates of both cell strains. We conclude that YPR128cp most likely mediates the transport of ATP across the peroxisomal membrane.Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of an increasing number of inherited diseases in man in which one or more peroxisomal functions are impaired (24,40,50). One of the main functions of peroxisomes is the degradation of fatty acids. In vertebrates, this takes place not only in peroxisomes but also in mitochondria. Long-chain fatty acids (LCFAs) and medium-chain fatty acids (MCFAs) are oxidized in mitochondria, whereas very longchain fatty acids and certain branched-chain fatty acids are first shortened in peroxisomes and subsequently oxidized to completion in mitochondria. This and other metabolic functions of peroxisomes (30,40,50) imply the existence of transport proteins in the peroxisomal membrane to shuttle metabolites from the interior of peroxisomes to the cytosol and vice versa. Indeed, several reports have appeared indicating the existence of such carrier proteins (11,33,34,42,43,50).We and others have been using Saccharomyces cerevisiae as a model organism to study the functions of peroxisomal membrane proteins (PMPs) for a number of reasons. First, in contrast to mammalian cells, peroxisomes in yeast are the sole organelles in which -oxidation of fatty acids takes place (18). Second, S. cerevisiae is an easy organism to manipulate genetically, and its entire genome sequence is available to enable specific studies. Third, S. cerevisiae can use fatty acids as sole carbon source and therefore mutants disturbed in fatty acid -oxidation can be readily identified by their growth characteristics in media supplied with different fatt...