Three forms of cytochrome P-450 in rat liver microsomes (Comai, K. and Gaylor, J.L. (1973) J. Biol. Chem. 284, 4947-4955) were characterized as two types of P-450 in the membrane-bound state. One was reactive not only toward cyanide but also aniline, aminopyrine, and hexobarbital, and the other was less reactive toward cyanide and rather unreactive toward the drugs. Cyanide titrations of microsomes in the presence and absence of the drugs were performed spectrophotometrically for analysis of the interactions. The affinity of cyanide for the less reactive P-450 was about twenty times less than that for the reactive P-450. About 40% of P-450 in the microsomal membrane was reactive and the rest was the less reactive form. The reactive P-450 involved two forms having different affinities for cyanide. The ratio of their amounts was 1:2. Triton WR-1339 interfered with cyanide binding to the reactive P-450 mainly. The relative amount of each form of P-450 as well as the dissociation constant of cyanide could be estimated by the present method, and the modes of interaction of the membrane-bound P-450 with the drugs and exogenous ligands were deduced.