Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of obtaining energy by oxidizing ferrous iron or sulfur compounds such as metal sulfides. Some of the proteins involved in these oxidations have been described as forming part of the periplasm of this extremophile. The detailed study of the periplasmic components constitutes an important area to understand the physiology and environmental interactions of microorganisms. Proteomic analysis of the periplasmic fraction of A. ferrooxidans ATCC 23270 was performed by using high resolution linear ion trap-Fourier Transform mass spectrometry (LTQ-FT/MS). We identified a total of 131 proteins in the periplasm of the microorganism grown in thiosulfate. When possible, functional categories were assigned to the proteins and found 13.8% of transport and binding proteins, 14.6% of several kinds of cell envelope proteins, 10.8% in energy metabolism, 10% related to protein fate and folding, 10% of proteins with unknown functions and 26.1% of proteins without homologues in databases. These last proteins are most likely characteristic of A. ferrooxidans and may have important roles yet to be assigned. The majority of the periplasmic proteins from A. ferrooxidans were very basic compared with those of neutrophilic microorganisms such as Escherichia coli, suggesting a special adaptation of the chemolithoautotrophic bacterium to its very acidic environment. The high throughput proteomic approach used here not only helps to understand the physiology of this extreme acidophile but also offers an important contribution to the functional annotation for the available genomes of biomining microorganisms such as A. ferrooxidans for which no efficient genetic systems are available to disrupt genes by procedures such as homologous recombination.