The sequence of the nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome b6f complex of Chlamydomonas reinhardtii has been established. Comparison of genomic clones and amplified cDNA indicates that the petC gene is interrupted by four introns within the coding sequence of the mature protein. The nucleotide sequence predicts a precursor protein of 206 amino acid residues with a transit peptide of 29 amino acids. The transit peptide is shorter than that of higher plants and has a basic region typical for the transfer through the chloroplast envelope, but no hydrophobic segment at the C-terminal end as is found in proteins transferred through the thylakoid membrane. The mature protein shows a high degree of homology with that of higher plants and has an N-terminal hydrophobic segment as in other Rieske proteins. Biochemical data (Breyton, C., de Vitry, C., and Popot, J.-L. (1994) J. Biol. Chem. 269, 7597-7602) indicate that the chloroplast Rieske protein of C. reinhardtii is an extrinsic membrane protein. Therefore, this N-terminal hydrophobic segment is not a transmembrane segment but may act as an uncleaved N-terminal thylakoid membrane transfer signal sequence. There are other examples of uncleaved hydrophobic membrane transfer signal in secreted proteins, although these are rare.