Efficient bioleaching requires adequate access to oxygen to drive the biochemical reactions that underpin iron and sulfur oxidation and ultimately copper solubilization. However, microaerophilic or anaerobic conditions may occur in certain parts of the heap, especially in areas of intense microbial activity or in biofilms where oxygen gradients occur. Microaerophilic conditions have also been detected in pristine acidic environments and in abandoned bioleaching operations. An important microorganism in bioleaching at ambient temperatures is the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. In addition to its well established role in oxidative processes, it has been shown to be capable of reducing metals such as iron and sulfur, and the products of these reactions may promote passivation of mineral surfaces and impede efficient solubilization of copper. In an effort to advance our understanding of the genetic and physiological basis of anaerobic metabolism, gene clusters controlled by the master anaerobic transcriptional regulator FNR were predicted in the genome of A. ferrooxidans using bioinformatics techniques. These clusters were found to be associated with energy metabolism, nitrogen fixation and carbohydrate metabolism. The results not only support previous evidence for proposed anaerobic metabolic pathways but also identify new genetic components and pathways that may be important for anaerobic or microaerophilic growth of this microorganism.