Although liver perfusion in situ is a valuable technique for the study of hepatic metabolism, it has rarely been applied to the study of mice. This is primarily due to the lack of a simple, reproducible technique to cannulate the delicate portal vein and initiate perfusion without irreversible hepatic injury. Therefore, we developed a technique to overcome these obstacles and performed studies to assess the viability of the perfused murine liver. The inflow catheter assembly permits rapid, reproducible cannulation of the portal vein and protects the cannulated vein from the effects of subsequent manipulations. Serial assessments of perfusion pressure, flow rate, perfusate pH, oxygen consumption, and fluxes of alanine aminotransferase and potassium indicate that the murine liver remains viable during perfusion. Moreover, the light and electron microscopic appearance of perfused livers is indistinguishable from that of freshly excised control livers. This technique should facilitate perfusion studies of hepatic metabolism in murine strains with genetic homogeneity and defined genetic abnormalities of hepatic metabolism.