Among two hundred strains of iron-oxidizing bacteria isolated from the natural environment, only one, Thiobacillus ferrooxidans NASF-1, grew in an Fe2+ medium supplemented with 10 mu M of 2,4-dinitrophenol (2,4-DNP). In contrast, growth of T. ferrooxidans AP19-3, a strain representative of iron oxidizing bacteria sensitive to 2,4-DNP, was strongly inhibited by 2,4-DNP at 1.0 mu M , and completely repressed at 10 mu M. Strain NASF-1 was also resistant to an other uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP). The iron-oxidizing activities of strains AP19-3 and NASF-1 were respectively inhibited 69 and 23% by 10 mu M 2,4-DNP, while the (CO2)-C-14 uptake activities of the two strains were inhibited 31 and 5% by 2,4-DNP at 1.0 mu M, and 100 and 89% at 5.0 mu M. When intact cells of strain AP19-3 were incubated with Fe2+ and 1.0 mu M 2,4-DNP for 10 min at pH 3.0, 49 pmol 2,4-DNP per mg of protein was incorporated into the cells. In contrast, strain NASF-1 did not incorporate 2,4-DNP under the same conditions, suggesting that, compared with strain NASF-1, strain AP19-3 has a plasma membrane through which 2,4-DNP is more easily incorporated in the cytosol.