There is an increasing need for continuously monitoring changes in brain metabolism and neuronal activity, respectively. The aim is to improve our understanding of mechanisms involved in physiological as well as pathophysiological and behavioural responses and to characterise drug actions. Changes of NADH concentration in the brain can be regarded as an index of changes in mitochondrial activity, which is closely related to neuronal activity. During the last decade the determination of NADH fluorescence by laser-induced fluorescence spectroscopy has become a method of choice in the study of mitochondrial metabolism in neuroscience. By now, small optical probes, providing excellent temporal and spatial resolution and the development of reliable and robust laser-based fluorescence detectors allow a widespread use in preclinical research. Besides in vitro studies, especially the assessment of changes in the NADH fluorescence in vivo has been shown to provide valuable information on brain function. Several applications are given, ranging from studying drug action or the extent of brain lesion to the measurement the time course of NADH concentration in a brain region of an awake and behaving laboratory rat. Theoretical aspects, opportunities, and limitations that have to be considered during the implementation of fluorescence spectroscopy are described. It is concluded, that measurement of NADH fluorescence by laserinduced fluorescence spectroscopy is a suitable tool for investigation of functional processes in the brain. Distribution and intensity of the NADH fluorescence in coronal rat brain slices containing the hippocampus. The data represent the intensity of the NADH fluorescence in arbitrary units and are shown as averaged data (n=5) from each measuring point, respectively