The slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps: first reduction of cytochrome P-450 and then some changes in reduced state. The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423 nm and 429 nm, respectively, while that of azide had a peak at 445 nm and a trough at 404 nm. An essay method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducible pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425 nm.