As a first step to characterize the unknown functional properties of the human mitochondrial ADP/ATP carrier (AAC), we tried to express human type 1 AAC (hAAC1) in Saccharomyces cerevisiae. Expression of hAAC1 in yeast mitochondrial membrane was very low, although its transcript level was high. Its expression was improved greatly by replacement of its N-terminal region with the corresponding region of yeast type 2 AAC (yAAC2), as observed with the bovine type 1 AAC (bAAC1). This chimeric hAAC1 showed similar ADP transport activity to that of chimeric bAAC1, corresponding to the transport activity of bAAC1 in bovine heart mitochondria. These results suggested that the N-terminal region of yAAC2 is important for expression of the mammalian carriers in yeast mitochondria. Using the present expression system, studies on the functional properties of the human AAC isoforms in relation to their structures are now possible.