Thiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe2+ or sulphide as energy source. Growth curves for Thiobacillus ferrooxidans have been tested, which show lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Thiobacillus ferrooxidans cultivated with Fe2+ and from 4 to 12 days for Thiobacillus ferrooxidans cultivated with elemental sulphur. Differences of protein patterns of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately were investigated after cultivation at 30 degrees C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ ionization (MALDI)-Mass spectrometry and ESI-MS/MS. From the 17 identified protein spots, 11 spots were found more abundant when growing on elemental sulphur. By contrast 6 protein spots were found decreased at elemental cultivation condition. Among the proteins identified, cytochrome C have been previously identified as necessary elements of electron-transferring pathway for Thiobacillus ferrooxidans to oxidize Fe2+; ATP synthase alpha chain and beta are expressed increased when Thiobacillus ferrooxidans cultivated with Fe2+ as energy source. ATP synthase Beta chain is the catalytic subunit, and ATP synthase alpha chain is a regulatory subunit. The function of ATPase produces ATP from ADP in the presence of a proton gradient across the membrane.