Abstract This study compared the NADH- and NADPH-supported p-nitrophenetole (NP) O-deethylase, ethylmorphine (EM) O-deethylase and EM N-demethylase activities of rat hepatic microsomes with respect to dioxygen requirement, inhibition by carbon monoxide, inhibition by classical inhibitors of cytochrome P-450 systems, and the involvement of NADH-cytochrome b 5, cytochrome b 5 reductase and NADPH-cytochrome P-450 reductase. The results generated the following conclusions and speculations: (1) NADH- and NADPH-supported O-deethylations of NP involve different P-450 hemoproteins. This conclusion was based largely on the observations that (a) the NADPH-supported reaction was inhibited by carbon monoxide and cyanide (5mM), whereas the NADH-supported reaction was not: (b) the NADH-supported reaction required a relatively high pO 2 for maximal activity, whereas the NADPH-supported reaction did not, and (c) the NADPH-supported reaction was depressed in microsomes from rats that had been administered Co 2+, Mn 2+, allylisopropylacetamide (AIA) or polyriboinosinic acid · polyribocytidylic acid (poly IC), whereas the NADH-supported reaction was not. However, the NADH- and NADPH-supported reactions shared some common features: both were strongly inhibited by α-naphthoflavone and weakly inhibited by 2-diethylaminoethyl 2,2-diphenyl valerate HC1 (SKF 525-A). both were destroyed by linoleic acid hydroperoxide, and both were induced by 3-methylcholanthrene (MC) and phenobarbital. The use of antibodies against NADPH-cytochrome P-450 reductase, NADH-cytochrome b 5, reductase and cytochrome b 5 demonstrated that both the NADH-and the NADPH-supported reactions depend on established components of cytochrome P-450 systems. (2) The P-450 hemoproteins involved primarily in both the NADH- and NADPH-supported deethylation of NP are the P 1-450 type, i.e. they are markedly induced by MC and inhibited by α-napthoflavone. (3) The NADH- and NADPH-supported O-deethylations of NP involve separate electron transfer systems. This conclusion is based largely on the following observations: (a) the synergism of the reaction, observed under normal atmospheric conditions when both NADH and NADPH were present, was not seen when the pO 2 was restricted to 5%; (b) studies of the inhibitory effect of antibody against cytochrome b 5 showed that cytochrome b 5 was an absolute requirement of the NADH-supported reaction, but not of the NADPH-supported reaction; (c) studies that used antibody against NADPH-cytochrome P-450 reductase, the inhibitory effect of NADP +, and the treatment of microsomes with trypsin showed that NADPH-cytochrome P-450 reductase was not involved in the NADH-supported reaction; (d) the use of antibody against cytochrome b 5 showed that cytochrome b 5 was required for a variety of NADH-supported monooxygenase reactions but not for the same NADPH-supported reactions. It is postulated from these observations that the cytochrome P-450(s) involved in the NADH-supported reaction is primarily NADH-dependent. The possibility is considered that this cytochrome P-450 may not exist as such normally but may be created from a more conventional cytochrome P-450 when it combines with NP to form a modified cytochrome P-450 with diminished affinities for carbon monoxide and dioxygen. (4) NADH- and NADPH-supported O-deethylation of NP, O-deethylation of EM and N-demethylation of EM involve different cytochrome P-450 systems. This conclusion is based on the observation that none of the three reactions responded alike to all of a variety of conditions to which they were subjected.