Acidithiobacillus ferrooxidans is an acidophilic, chemolithotrophic bacterium that can gain energy and electrons by the oxidation of iron. PetI is an operon that encodes electron transport proteins involved in the reverse flow of electrons from iron to the NAD complex during iron oxidation. Although a substantial body of evidence describes the components and pathways for iron oxidation, little is known about their regulation. It is proposed that environmental iron concentrations and oxygen levels could influence the ability of A. ferrooxidans to oxidize iron as an energy source. We report initial investigators into the possibility that the expression of the PetI operon is regulated, inter alia, by the master transcription factor controlling iron uptake and homeostasis (Fur) and anaerobic metabolism (FNR). Potential DNA binding sites (boxes) for both regulators were predicted in the region of the PetI operon promoter and the binding of Fur to its cognate box was demonstrated experimentally. This represents the first attempt to unravel the mechanisms involved in the regulation of iron oxidation.