A microscopic reflectance spectrophotometer was constructed to obtain the spectra of single pial vessels and of a region containing only capillaries (capillary region). The difference in the oxygen saturation (SO2) of hemoglobin between the regional arteriole and venule [R(A - V)] and that between the regional arteriole or capillaries [R(A - C)] were calculated. The reduction of cytochrome aa3 was also estimated in the capillary region. This method was applied to the brain surface of spontaneously breathing rats subjected to hypoxic and anemic hypoxia. On decreasing the inhaled O2 from 100 to 15%, elevation of R(A - V) and R(A - C) with slight arteriolar dilatation (though statistically not significant) was observed. Below 10% O2 (especially at 4 and 3% O2), the R(A - V) and R(A - C) decreased in spite of significant arteriolar dilatation with progressive reduction of cytochrome aa3, indicating suppression of oxygen transport to mitochondria. In the case of hemodilution down to 37% hematocrit (Ht), elevation of R(A - V) and R(A - C) occurred with a slight tendency toward arteriolar dilatation. Below 32% Ht, the R(A - V) decreased but the R(A - C) remained steady, while reduction of cytochrome aa3 progressed. Altogether, the SO2 in the capillary region decreased and the reduction of cytochrome aa3 progressed with the decline of arteriolar O2 supply in both hypoxic and anemic hypoxia.