The interactions between purified microsomal cytochrome P-450 and cytochrome b 5 has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital ( P-450 LM2) and β-naphthoflavone ( P-450 LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b 5 are mainly distributed in the polyethylene glycol-rich top phase ( K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450 LM with cytochrome b 5 changes the partition coefficients of both P-450 LM and cytochrome b 5 indicating that molecular interaction between P-450 LM and cytochrome b 5 occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450 LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b 5, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b 5 decreases the K m's of both substrates for P-450 LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b 5 plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b 5 are catalytically competent in the electron transport reactions.