The significance of normal mitochondrial function in cellular energy homeostasis as well as its involvement in acute and chronic neurodegenerative disease was reviewed recently (Nicholls & Budd. Physiol Rev. 80: 315-360, 2000). Nevertheless, monitoring of mitochondrial function in vivo and real time mode was not used by many investigators and is very rare in clinical practice. The main principle tool available for the evaluation of mitochondrial function is the monitoring of NADH fluorescence. In order to interpret correctly the changes in NADH redox state in vivo, it is necessary to correlate this signal to other parameters, reflecting O2 supply to the brain. Therefore, we have developed and applied a multiparametric optical monitoring system, by which microcirculatory blood flow and hemoglobin oxygenation is measured, together with mitochondrial NADH fluorescence. Since the calibration of these signals is not in absolute units, the simultaneous monitoring provide a practical tool for the interpretation of brain functional state under various pathophysiological conditions. The monitoring system combines a time-sharing fluorometer-reflectometer for the measurement of NADH fluorescence and hemoglobin oxygenation as well as a laser Doppler flowmeter for the recording of microcirculatory blood flow. A combined fiber optic probe was located on the surface of the brain using a skull cemented cannula. Rats and gerbils were exposed to anoxia, ischemia and spreading depression and the functional state of the brain was evaluated. The results showed a clear correlation between O2 supply/demand as well as, energy balance under the various pathophysiological conditions. This monitoring approach could be adapted to clinical monitoring of tissue vitality.