The near infrared absorption spectra was measured with the transmitted light through rat brain under the various condition. The absorbance changes below 780 nm were attributable to hemoglobin (Hb) in the brain tissue, whereas those above 780 nm were associated with both Hb and cytochrome oxidase. To eliminate possible interference from cyt. oxidase, two wavelengths, 750 nm and 780 nm, were used to measure Hb oxygenation in the tissue. The absorbance changes in human blood cell suspensions were measured with changes in hematocrit values in the optical cuvette. At two wavelengths 750 nm and 780 nm, there was a linear relationship between absorbance changes and hematocrit values. Through these in vitro studies, the following equation (1) and (2) were obtained to monitor quantitatively the changes of oxy-Hb content (delta Hb O2) and total-Hb content (delta Hb Vol.) in the living tissue. These are (1) delta Hb O2 = -1.15 delta A 750 + 1.39 delta A 780, (2) delta Hb Vol. = -0.29 delta A 750 + 0.59 delta A 780. The studies using these equations showed that the oxy-Hb content in the brain was decreased as the O2 concentration in inspired gas was lowered with a half of Hb deoxygenated at 7% O2. The reliability of these equations was examined under the various conditions in situ such as CO2 inhalation, intravenous injection of Ca2+-blocker nicardipine, hemorrhage and retransfusion. These results confirmed that these equations derived from in vitro studies, were successfully applied to the in situ measurements of the oxygenation state of Hb in the living tissues.