The sulfur metabolism of Thiobacillus ferrooxidans, Ferrobacillus ferrooxidans, and Ferrobacillus sulfooxidans was found to differ in the rate of utilization of thiosulfate and tetrathionate, with tetrathionate showing the most significant difference between the three organisms. The apparent K m values of the three organisms ranged from 2–5 × 10 −2 M for T. ferrooxidans and F. sulfooxidans to 5–7 × 10 −3 M for F. ferrooxidans. Increasing the temperature caused a greater effect on thiosulfate oxidation by F. ferrooxidans than with the other two as shown by the apparent E a values (E a ′ for F. ferrooxidans, 18 000; F. sulfooxidans, 11 400; T. ferrooxidans, 11 000).The initial pH of the medium determined the rate of thiosulfate oxidation. If the initial pH was 4.0–4.5, a sharp break in oxygen uptake was shown by T. ferrooxidans and F. sulfooxidans after 20–30 min, but not with F. ferrooxidans.When the disappearance of thiosulfate and tetrathionate from the medium was used as an indicator of the affinity of the three test organisms for the sulfur compounds, F. ferrooxidans demonstrated the highest net loss of both compounds from the medium.