We developed a method for quantitating absolute value of Hb oxygenation and that of redox state of cyt. aa3 on the basis of multiple regression analysis of near-infrared spectrophotometric data. Flattening of the spectrum of Hb in both RBC suspension and the brain in situ was observed. This phenomenon was explained by localization of Hb within RBC for in vitro flattening, and by localization of RBC within the vessels as well as deformation of RBC in the microcirculation in vivo. Under resting air-breathing conditions, So2 and redox state of cyt. aa3 were estimated to be 50-70% and over 90%, respectively. Increase in FIO2 up to 3 ATA O2 caused stepwise increase in So2, whereas cyt. aa3 was fully oxidized when the animal inspired O2 under 1 ATA. Problems which must be solved for more accurate estimation of absolute values of So2 and redox state of cyt. aa3 were described and discussed.