The sensitivity of the brain to cyanide-induced histotoxic hypoxia and the protective effects of known cyanide antagonists, have been assessed in vivo by reflectance spectrophotometry. Cyanide-related changes in cytochrome a,a3 (cytochrome c oxidase) oxidation-reduction (redox) state, tissue hemoglobin saturation, and local blood volume were continuously monitored in cerebral cortex of rats. Noncumulative, dose-dependent inhibition of the in situ mitochondrial respiratory chain was evaluated directly by measuring increases in reduction levels of the terminal oxidase. These transient cytochrome a,a3 reductions were accompanied by increases in regional cerebral hemoglobin saturation and blood volume. Cytochrome redox responses were not altered either in magnitude or kinetics by hyperoxia; however, the cyanide-cytochrome dose-response curve was greatly shifted to the right by pretreatment with sodium nitrite, and the recovery rate of cytochrome a,a3 from cyanide-induced reduction was enhanced fourfold by pretreatment with sodium thiosulfate.