The recent availability of an incomplete genomic sequence from Acidithiobacillus ferrooxidans allowed us to continue and strengthen the demanding task of investigating the proteome and its functional implications in this extremophilic microorganism. The proteins of At. ferrooxidans were separated by two-dimensional polyacrylamide gel electrophoresis and their levels of synthesis and the microsequencing of their N-terminal end amino acids were determined. To link the 2D gel spots of interest with the genes that encodes them, we studied the global changes in gene expression of At. ferrooxidans when the bacterium was confronted with phosphate starvation. By comparing the amino acid sequences of the proteins whose synthesis was induced or repressed under these conditions, with the available At. ferrooxidans genomic database, we found several putative genes whose expression may be related to phosphate starvation. Analysis of the genome DNA sequences upstream and downstream of these genes showed us details of the structure of putative operons present in At. ferrooxidans, strongly suggesting the existence of a Pho regulon containing the putative genes phoB, phoR, pstS, pstC,pstA, pYtB, phoU, ppx and ppk. Some differences were seen in the organization of the genes in the possible Pho regulon of At. ferrooxidans when compared with the Pho operons from other microorganisms. This was specially evident in the organization of the genes involved in polyphosphate metabolism (ppk and ppx). Regulation of phosphate metabolism is of particular relevance when At, ferrooxidans grows in the presence of arsenopyrites, which release arsenate, a structural analog of phosphate. Structural comparison between the specific phosphate-binding protein PstS from Escherichia coli and the corresponding At. ferrooxidans homolog showed that both proteins are highly conserved, including the phosphate/arsenate binding site, which shares seven of the eight amino acid residues necessary for the hydrogen bonding to the four oxygens of phosphate.