We have previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 system, through the hepatic deletion of either cytochrome P450 oxidoreductase (POR; HRN line) or cytochrome b5 (Cyb5; HBN line). However, HRN mice still exhibit low levels of mono-oxygenase activity, in spite of the absence of detectable reductase protein. To investigate whether this is because cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the P450 system, we have crossed HRN with HBN mice to generate a line lacking hepatic expression of both electron donors (HBRN). HBRN mice exhibited exacerbation of the phenotypic characteristics of the HRN line - liver enlargement, hepatosteatosis and increased expression of certain cytochrome P450s. Also, drug metabolising activities in vitro were further reduced relative to the HRN model, in some cases to undetectable levels. Pharmacokinetic studies in vivo demonstrated that midazolam half-life, Cmax and area under the concentration-time curve (AUC) were increased, and clearance was decreased, to a greater extent in the HBRN line than in either the HBN or HRN model. Microsomal incubations using NADPH concentrations below the apparent Km of cytochrome b5 reductase, but well above that for POR, led to the virtual elimination of 7-benzyloxyquinoline turnover in HRN samples. These data provide strong evidence that cytochrome b5/cytochrome b5 reductase can act as a sole electron donors to the cytochrome P450 system in vitro and in vivo.