When Escherichia coli strain B/r is exposed to 10 to 20 μg of nitrofurazone per ml, mutants with roughly threefold resistance are obtained. Treatment of these mutants with higher concentrations of nitrofurazone yields strains with six- to seven-fold resistance over strain B/r. Each of these steps toward nitrofurazone resistance is accompanied by loss of soluble nitrofurazone reductase activity. When sensitive bacteria are exposed to labeled nitrofurazone or labeled 2-nitrofuran, a considerable amount of radioactivity becomes bound to the cold trichloroacetic acid-insoluble fraction. Very little activity becomes bound in the mutants with six- to seven-fold resistance; mutants with intermediate resistance show intermediate levels of binding. Partially purified nitrofurazone reductase preparations catalyze the conversion of nitrofurazone to compounds which bind to protein and are not removed by prolonged dialysis against 8 m urea or by cold acid. Nitrofurazone reduced by xanthine oxidase or electrolytically reduced also yields compounds which react with protein to form stable derivatives.