Acidithiobacillus ferrooxidans is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidate fur(AF) (Ferric Uptake Regulator) gene was identified in the A. ferrooxidans ATCC 23270 genome. Fur(AF) has significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity to Escherichia coli Fur antiserum. The fur(AF) gene is able to complement fur deficiency in E. coli in an iron-responsive manner. Fur(AF) is also able to bind specifically to E. coli Fur regulatory regions (Fur boxes) and to a candidate Fur box from A. ferrooxidans, as judged by electrophoretic mobility shift assays. Fur(AF) represses gene expression from E. coli Fur-responsive promoters fiu and fhuF when expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.