The cyanide-sensitive factor (CSF), the terminal oxidase of the stearoyl-CoA desaturase system, was solubilized with detergents from rat liver microsomes and separated by DEAE-Sephadex chromatography from NADH-cytochrome b₅ reductase [EC 1. 6. 2. 2] (fp₁) and cytochrome b₅. An NADH-dependent stearoyl-CoA desaturase activity could be reconstituted by mixing the CSF fraction with detergent-solubilized fp₁ (d-fp₁) and detergent-solubilized cytochrome b₅ (d-b₅), both purified from rabbit liver microsomes. The reconstituted activity was similar to that of liver microsomes in pH optimum and behavior towards inhibitors. Omission of any one of the three components led to almost complete loss of the reconstituted activity. Trypsin-solu-bilized cytochrome b₅ (t-b₅) was only negligibly active in the reconstitution. When combined with the CSF fraction, lysosome-solubilized fp₁ (1-fp₁), but not d-fp₁, catalyzed the desaturation even in the absence of cytochrome b₅. This suggested that 1-fp₁, which has been shown to be a proteolytically modified product of d-fp₁, had acquired an artificial activity to react directly with CSF. The isolated CSF fraction was colorless, containing no heme and cytochromes, but was rich in phospholipids. It is concluded that cytochrome b₅, in addition to fp₁ and CSF, is obligatorily involved in the microsomal desaturase system and that CSF is probably not a hemoprotein.