From several presumably pure cultures of Thiobacillus ferrooxidans, we isolated a pair of stable phenotypes. One was a strict autotroph utilizing sulfur or ferrous iron as the energy source and unable to utilize glucose; the other phenotype was an acidophilic obligate heterotroph capable of utilizing glucose but not sulfur or ferrous iron. The acidophilic obligate heterotroph not only was encountered in cultures of T. ferrooxidans, but also was isolated with glucose-mineral salts medium, pH 2.0, directly from coal refuse. By means of deoxyribonucleic acid homology, we have demonstrated that the acidophilic heterotrophs are of a different genotype from T. ferrooxidans, not closely related to this species; we have shown also that the acidophilic obligate heterotrophs, regardless of their source of isolation, are related to each other. Therefore, cultures of T. ferrooxidans reported capable of utilizing organic compounds should be carefully examined for contamination. The acidophilic heterotrophs isolated by us are different from T. acidophilis, which is also associated with T. ferrooxidans but is facultative, utilizing both glucose and elemental sulfur as energy sources. Since they are so common and tenacious in T. ferrooxidans cultures, the heterotrophs must be associated with T. ferrooxidans in the natural habitat.