Aromatic nitro compounds interact with sodium dithionite-reduced microsomes to generate difference spectra characterized by a maximum at 400 nm and minima at 440 and 528 nm. Spectral binding constants of approximately 1 mM were calculated for ferrohemochromes in which nitrobenzene, p-nitrobenzoate, 2-nitrofluorene, and 2-nitronaphthalene served as ligands. Nitro binding affinity was increased approximately 3-fold by pretreatment of animals with phenobarbital (KS congruent to 0.3 mM). The interaction of nitro compounds with reduced microsomes from 3-methylcholanthrene-pretreated animals, however, failed to give discernable difference spectra. There is apparent mutual inhibition of binding of carbon monoxide, metyrapone, and nitro compounds with reduced cytochrome P-450, since addition of metyrapone or carbon monoxide to reference and sample cuvettes does not alter qualitative aspects of the nitro binding spectra, but only its magnitude. From the relative KS values, it is concluded that metyrapone and carbon monoxide interact with reduced cytochrome P-450 more tenaciously than nitro compounds.