When Thiobacillus ferrooxidans was grown on ferrous iron and chalcopyrite (CuFeS2) in excess of 96% of the bacterial population was associated with the insoluble material. When sulfur was the substrate 77% of the bacteria were so associated. This necessitated consideration of the complete growth system to obtain accurate growth curves. By using total bacterial nitrogen as the measure of growth, it was shown that T. ferrooxidans had a minimum generation time of 6.5 to 10 hours on a ferrous iron substrate, 7 to 8 days on a sulfur substrate, and 14 to 17 hours on a chalcopyrite substrate. The pH range for growth was dependent on the substrate used.