Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.