The role of cytochrome b5 in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion (ADME) and toxicity are controversial. In order to resolve this issue we have previously generated the hepatic cytochrome b5 null (HBN) mouse. In vitro and in vivo drug metabolism studies carried out in these mice confirm an unequivocal role for cytochrome b5 in hepatic P450 drug metabolism. To extend our knowledge of the in vivo role of cytochrome b5, particularly in extra-hepatic drug metabolism, we have now generated a complete knockout mouse model for this enzyme. Cytochrome b5 complete null (BCN) mice are fertile, grow normally and display no anatomical abnormalities. However, unlike hepatic deletion of cytochrome b5, global deletion resulted in significant changes in P450 expression levels in various tissues, particularly the liver. In vitro studies using a range of substrates for different P450 isozymes showed that global deletion of cytochrome b5 led to significant changes in the NADPH-dependent metabolism of these substrates by liver and kidney microsomes. These changes were also reflected in the in vivo elimination of the orally administered drugs, midazolam, phenacetin and metoprolol, the pharmacokinetics of which were markedly altered in BCN compared to wild-type mice. In addition, their elimination profiles were similar to those observed in HBN mice. These data indicate that cytochrome b5 plays a major role in both hepatic and extra-hepatic drug metabolism.