We have examined the effects of uncoupling on electron transfer reactions and cell bioenergetics in rat brain in situ. The redox reactions of cyt aa₃, cyt c and cyt b as well as the levels of phosphocreatine/inorganic phosphate ratio (P_Cr/P_i) were monitored in isolated perfused rat head, using optical and [³¹P]NMR techniques, respectively. In the first series of experiments anesthetized (pentobarbital) and mechanically ventilated rats underwent bilateral carotid arterial cannulation. The head (skull intact, muscle removed) was perfused with a perflurochemical solution (FC-43), and the animal was then decapitated. By means of reflectance spectrophotometry, the redox reactions of cyt aa₃, c and b were monitored before and after perfusing the head with uncoupler 2,4-dinitrophenol (DNP) as well as after complete anoxia. The second series of experiments was performed using [³¹P]NMR spectroscopy (24.33 MHz; 10.25 in. magnet). P_Cr/P_i ratios were first determined for living rats (in vivo) before and after exchange transfusion with FC-43 (Hct < 0.5%), and then in isolated perfused head (in situ) before and after 2,4-DNP addition. Reduction of cyt aa₃ (32 ± 1.4%,mean±S.E.M.), cyt c (29 ± 6%), and cyt b (19 ± 2%) was induced by addition of 2,4-DNP. Compared to in vivo aerobic metabolism with a P_Cr/P_i ratio of 3.0 – 5.0, the value in the uncoupled state in situ was < 0.1. We conclude that in contrast to in vitro, uncoupling of oxidative phosphorylation induces reduction of intramitochondrial cytochromes in situ. It seems that under these experimental conditions, the marked increase in electron transport due to stimulated respiration induced by the uncoupler far exceeds the available oxygen, simulating a state 3 (resting) to a state 5 (hypoxic) transition. The dramatic fall in P_Cr/P_i ratio indicates energy coupling loss and supports the hypothesis that hyperactive respiration causes tissue hypoxia resulting in reduced cytochromes. Combining optical and NMR techniques will permit assessment of oxygen delivery to tissue, the function of mitochondria, and the coupling of bioenergetic processes in vivo.