Higher plant photosystem II preparations contain a 4.1 kDa polypeptide (subunit X) associated with the oxygen-evolving core complex. We describe the isolation of a cDNA encoding PS II-X from Arabidopsis thaliana, in which the C-terminal region is highly homologous to partially sequenced PS II-X from wheat and spinach. The mature protein of 42 residues is preceded by a 74-residue, bipartite presequence similar to those involved in the targeting of nuclear-encoded thylakoid lumen proteins, although hydrophobicity analysis indicates the presence of a single transmembrane span in the mature protein. Moreover, import of pre-PS II-X into the thylakoid membrane of isolated chloroplasts is unaffected by inhibitors of either the Sec- or ΔpH-dependent thylakoidal protein translocases, suggesting a spontaneous insertion mechanism. PS II-X appears to be encoded as a mature protein by the plastid genome in the chlorophyll a+c-containing alga, Odontella sinensis. We thus propose that the thylakoid transfer signal of Arabidopsis pre-PS II-X represents a recent acquisition, in phylogenetic terms, compared with signals of Sec-dependent lumenal proteins.