Using a spectrophotometer connected to a microcomputer, the carbon monoxide difference spectrum of renal microsomal dithionite-reduced cytochrome P-450 was measured avoiding the effects of the contaminating mitochondrial cytochromes and hemoglobin by subtracting the CO difference spectrum of a succinate-treated microsomal suspension from that of a dithionite-treated one. By this method, we quantitatively determined the microsomal cytochrome P-450 in kidneys from various species. The absorption peak of the renal microsomal cytochrome P-450 was about 452 nm in rats and about 450 nm in mice, hamsters, rabbits, guinea pigs, dogs, and pigs. Compared with the renal cytochrome P-450 contents in rats, the contents on a g tissue basis were greater in pigs, dogs, and mice and were about the same in hamsters and rabbits. The renal cytochrome P-450 contents in guinea pigs were less than those in rats.