We previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 (P450) system, through the hepatic deletion of either cytochrome P450 oxidoreductase [POR; HRN (hepatic reductase null) line] or cytochrome b₅ [HBN (hepatic cytochrome b₅ null) 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 the sole electron donor to the P450 system, we 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 P450s. Also, drug metabolizing 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, C_max, and area under the concentration-time curve 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 K_m 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 donor to the P450 system in vitro and in vivo.