Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [35S]cysteine, in which [35S]sulfide and iron are incorporated into Fd to build up the 35S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [35S]sulfide or iron, or in Fe-S cluster formation itself. [35S]Sulfide was liberated from [35S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [35S]cysteine occurred before the formation of the 35S-labeled Fe-S cluster, and the amount of radioactivity in [35S]sulfide was greater than that in 35S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na2S) inhibited competitively formation of the 35S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [35S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.