Liver microsomes from phenobarbital-pretreated rabbits were digested with Nagarse (Bacillus subtilis proteinase) anaerobically in the presence of 25% glycerol at 0°C for 15 hr. Cytochrome b6 and NADPH-specific flavoprotein were almost completely solubilized by this treatment, but P-450, the other microsomal hemoprotein, was quantitatively retained in the microsomal particles without appreciable conversion to the modified P-420 state. The particles thus obtained (P-450 particles) containing P-450 as the sole protoheme constituent were suitable for measurements of absolute spectra of P-450. Using the preparation, bleached with hydrogen peroxide, as a turbidity control, the spectra of oxidized and dithionite-reduced P-450 particles were measured. The oxidized spectrum, having peaks at 360, 416, 535, 570 and 650 mμ, was not very atypical for protoheme-containing proteins. But the reduced spectrum, with absorption maxima at 412 and 555 mμ, was anomalous for hemoproteins. The spectrum obtained on addition of CO to the reduced sample showed absorption peaks at 449 and 555 mμ. Although the CO spectrum possessed a shoulder at 423 mμ, evidence was obtained to indicate that this was due to small amounts of P-420 and cytochrome b6 remaining in the preparation. Unusual interactions of ethyl isocyanide with both oxidized and reduced P-450, previously observed by difference spectrophotometry of intact microsomes, could be confirmed by measuring the absolute spectra of P-450 particles.