To quantify the changes in oxygen saturation of hemoglobin in the liver in hypoxia and liver transplantation, we applied a novel method using near-infrared spectroscopy. Instead of the conventional two-wave-length method, we obtained near-infrared data from a wide spectral range of 700 nm to 1,000 nm with continuous-wave spectroscopy. To correct the flattened spectral shape caused by photon scattering in living tissue, we then applied an equation taking into account the relationship between absorber concentration and actual absorption in scattering materials as assessed with time-resolved spectroscopy. Hepatic hemoglobin oxygen saturation was calculated with multicomponent analysis. In room air, hepatic hemoglobin oxygen saturation of rabbit was calculated as 57.4% +/- 2.5% (mean +/- S.E.M., n = 7). Arterial, portal and hepatic venous hemoglobin oxygen saturation were simultaneously measured as 97.5% +/- 0.7%, 77.1% +/- 3.4% and 55.5% +/- 4.6%, respectively. The changes in hepatic hemoglobin oxygen saturation seen in graded hypoxia were also close to those in hepatic venous hemoglobin oxygen saturation, suggesting that the average oxygenation state of sinusoidal blood approximates that in the central vein. We tested the clinical applicability of this method in a case of liver transplantation. It was determined that the hepatic hemoglobin oxygen saturation of the graft liver was heterogeneously distributed and that the initially low level of hepatic hemoglobin oxygen saturation was increased by the ligation of portal-systemic shunts.