Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans. Pyrite (FeS2) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe2+), elemental sulphur (S0) or FeS2. Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe2+ and S0 oxidation, NaN3 and NEM, respectively, partially abolished FeS2 oxidation. b. A b-type cytochrome was detectable in FeS2- and S0-grown cells but not in Fe2+-grown cells. c. FeS2 and S0 reduced b-type cytochromes in whole cells grown on S0. d. CO2 fixation at pH 4.0 per mole of oxygen consumed was the highest with S0, lowest with Fe2+ and medium with FeS2 as substrate. e. Bacterial Fe2+ oxidation was found to be negligible at pH 5.0 whereas both FeS2 and S0 oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe3+ was not affected by separation of pyrite and bacteria. Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.