Bloodless, anesthetized Sprague-Dawley rats produced by perfluorochemical exchange transfusion and splenectomy were used to obtain cerebral mitochondrial difference spectra directly through the skull by both reflectance and transmission spectrophotometry. Reflectance peaks were about one-third in amplitude compared to transmission peaks, however percentage reduction levels of cytochrome a, a3 in animals breathing 100% oxygen were comparable. Cytochrome a, a3 was 13-14% reduced under these conditions as determined by the ratio of the optical density difference at 605 minus 620 nm to the optical density difference at the same wavelength pair with maximal cytochrome a, a3 oxidation during respiration on 95% oxygen + 5% carbon dioxide at 3 and 4 atm. absolute pressure.