High-potential iron-sulfur protein (HiPIP) has been proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, which contains an [Fe4S4] cluster. We report here the assembly of an [Fe4S4] cluster in HiPIP from A. ferrooxidans ATCC 23270 in vitro in the presence of Fe2+ and sulfide. The spectra and matrix-assisted laser desorption ionization-time of flight mass spectrometry results of holoHiPIP confirmed that the iron-sulfur cluster was correctly assembled into the protein.