The reconstitution of the iron-sulfur cluster of spinach ferredoxin was examined in vitro using low and high molecular bound sulfur components as sulfur donors. Bound sulfur was rapidly converted to acid-labile sulfur to form an iron-sulfur center in the presence of dihydrolipoate and iron. Reconstitution yields of above 95% were obtained with cystine trisulfide (CT, 0.25 mM) and sulfur-bound albumin (SBA, 1.0 mM) at 37 degrees C, pH 7.3, following 60 min incubation. Spectroscopic features and biological activity of the reconstituted ferredoxin were identical to those of the native holo-protein. The acid-labile sulfur content found in the isolated reconstituted ferredoxin was 2 atoms/mol protein, similar to the theoretical value. A possible role for bound sulfur in mammalian cells is indicated and discussed.