The unusual difference spectrum appearing on combination of ethyl isocyanide with reduced P-450 in dithionite-treated liver microsomes was profoundly influenced by pH. Of the two Soret bands in the spectrum, the peak at 455 mμi was intensified progressively as pH was increased, and this was accompanied by a corresponding decrease in the intensity of another peak at 430 mμz. Both peaks were competitively affected by CO to the same extent at any pH value and ethyl isocyanide concentration tested. The intensities of both peaks were similarly dependent on the ethyl isocyanide concentration, and the apparent dissociation constant determined from this dependence was 4.1 μM regardless of the pH employed. Interactions of aniline and pyridine with reduced P-450 also brought about spectral changes having two Soret bands. These spectra were also affected by pH in a similar way to that observed in the ethyl isocyanide interaction. It was suggested that reduced P-450, when in combination with lipophilic ligands such as ethyl isocyanide, aniline and pyridine, exists in two interconvertible states which are in a pH-dependent equilibrium. On combination with ethyl isocyanide, reduced P-420, the modified product of P-450, gave rise to only one Soret band, which was not affected by pH. The effect of the ethyl isocyanide concentration on this Soret band was, however, somewhat unusual.