The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, ''transit complex'', and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.The insertion of proteins into membranes is a fundamental process essential for the vitality of all organisms. The paradigm for this process is the targeting mediated by signal recognition particle (SRP), a cytoplasmic ribonucleoprotein. In prokaryotes, the SRP-RNA binds a single 54-kDa-polypeptide subunit, while in eukaryotes, five additional subunits are bound. One of the distinctive features of this targeting mechanism is cotranslational protein insertion. The synthesis of hydrophobic protein domains at the membrane circumvents potential protein folding problems that might otherwise occur in an aqueous environment. However not all hydrophobic proteins are targeted cotranslationally. For example, the major proteins of the thylakoid membrane, the light harvesting chlorophyll proteins (LHCP), are targeted by a posttranslational mechanism. LHCP form a large family of related proteins that have three to four transmembrane domains. They are synthesized in the cytoplasm, and are targeted to the thylakoid membrane through three aqueous compartments: the cytoplasm, the inter-envelope space, and the stroma (1, 2). The factors that mediate the posttranslational targeting of members of the LHCP family have not been definitively identified.The LHCP are inserted into the thylakoid membrane in a reaction requiring GTP and stroma (3-5). It was found previously that a stromal factor binds LHCP to form a soluble intermediate, designated transit complex, that maintains the solubility of these proteins as they are transported through the stroma (6, 7). The transit complex is the only soluble form of LHCP that accumulates when ...