Protein cross-linking studies with a thylakoid membrane translocation intermediate were used to demonstrate that chloroplast SecA functions as a membrane-associated component of the Sec-like ATP-dependent protein translocase of pea chloroplasts. In assays with isolated thylakoids, it was observed that translocation of the 33-kDa protein of the oxygen-evolving complex of photosystem II (OE33) decreased when the ATP concentration was low, and that the protein accumulated as a bound precursor. The bound precursor was able to be translocated into the lumen when the ATP concentration was raised, indicating that the precursor was bound to the translocation apparatus. Inclusion of apyrase in the import reaction prevented translocation but did not affect precursor binding to the membrane. When this translocation intermediate was treated with the cross-linking agent disuccinimidyl suberate, a single predominant cross-linked product of 120 kDa was produced. This conjugate could be immunoprecipitated with antibodies to pea chloroplast SecA, identifying the cross-linking partner as SecA. This provides direct evidence for a functional interaction between a thylakoid precursor protein and a component of the thylakoid protein-translocation apparatus.