1. Thus far metabolic processes in the intact animal (or man) have been studied either by the analysis of body fluids, of biopsies, of tissue obtained post mortem or by techniques, requiring dedicated and expensive equipment (such as positron emission tomography or magnetic resonance spectroscopy). 2. Here we describe a relatively simple and inexpensive technique, that can be applied in vivo to study metabolism in brain regions and muscle in the freely moving rat and in human peripheral tissue. 3. The method is based on microdialysis allowing continuous sampling from the extracellular space, the enzymatic conversion of lactate and the on-line detection of fluorescent NADH. 4. Examples of the application of our technique include the monitoring of lactate efflux from various brain regions of behaving animals under a variety of stress exposures, during ischemia or hypoxia and drug treatments. 5. The results indicate that in brain lactate is not exclusively formed under hypoxia and that neuronal activation leads also to lactate formation, possibly due to the compartmentation of both the involved enzymes and the energy metabolism. 6. The increase of lactate formation in contracting or ischemic muscle or during exercise could also be followed on-line in the rat, suggesting that our approach allows the continuous monitoring of anaerobic metabolism in man e.g. during traumatic or arteriosclerotic limb ischemia or lactic acidosis in shock states. 7. The principle of our approach can easily be adapted to other metabolites, thus enabling to monitor other metabolic pathways in vivo as well.