GENBANK/o acid sequence of the rat carnitine carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions with primers and probes based on amino acid sequence information, obtained from the direct sequencing of internal peptides of the purified carnitine carrier protein from rat. The protein sequence of the carrier, including the initiator methionine, has a length of 301 amino acids. The mature protein has a modified alpha-amino group, although the nature of this modification and the precise position of the N-terminal residue have not been ascertained. Analysis of the carnitine carrier sequence shows that the protein contains a 3-fold repeated sequence about 100 amino acids in length. Dot plot comparisons and sequence alignment demonstrate that these repeated domains are related to each other and also to the repeats of similar length that are present in the other mitochondrial carrier proteins sequenced so far. The hydropathy analysis of the carnitine carrier supports the view that the domains are folded into similar structural motifs, consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Southern blotting experiments suggest that both the human and the rat genomes contain single genes for the carnitine carrier. These studies provide the primary structure of the mitochondrial carnitine carrier protein and allow us to identify this metabolically important transporter as a member of the mitochondrial carrier family, and the sixth of the members whose biochemical function has already been identified.