By measuring precise NIR spectra of anesthetized rat head, we determined principal components included in changes of the NIR spectra caused by acute hypoxia. There was apparent delay in reduction of cyt. aa3 as compared with Hb deoxygenation in the hypoxic period. With regard to the eigenvalues determined by principal component analysis, principal components in the NIR spectral changes caused by acute hypoxia were Hb and cyt. aa3, and contribution of the remaining biological materials to the changes in NIR spectra caused by acute hypoxia was considered to be negligible in quantitative multivariate analysis of Hb and cyt. aa3 in situ.