Several cDNA clones encoding subunit XI of photosystem I reaction center (PSI-L) have been isolated from two lambda gt11 expression libraries based on polyadenylated RNA of spinach seedlings illuminated for 4 and 16 h, respectively. The precursor polypeptide made from these recombinant DNAs in vitro can be efficiently imported into isolated spinach chloroplasts. It is correctly processed to the size of the authentic polypeptide and integrates into the photosystem I assembly. The 834 nucleotide sequence of the longest cDNA insert encodes a precursor polypeptide of 24 kDa (216 residues) and a mature protein of probably 18.8 kDa (169 residues). Hydropathy analysis suggests that the polypeptide contains two transmembrane segments. The protein appears to originate in a single-copy gene in spinach and to be decoded from RNA species of ca. 900 bases.