During the last 5 to 10 years, the microdialysis technique has been used to explore neurotransmitter release during exercise. Microdialysis can collect virtually any substance from the brains of freely moving animals with a limited amount of tissue trauma. It allows the measurement of local neurotransmitter release in combination with ongoing behavioural changes such as exercise. Several groups examined the effect of treadmill running on extracellular neurotransmitter levels. Microdialysis probes were implanted in different brain areas to monitor diverse aspects of locomotion (striatum, hippocampus, nucleus accumbens, frontal cortex, spinal cord), food reward (hypothalamus, hippocampus, cerebral cortex), thermoregulation (hypothalamus). Some studies combined microdialysis with running on a treadmill to evaluate motor deficit and improvement following dopaminergic grafts in 6-hydroxydopamine lesioned rats, or combined proton nuclear magnetic resonance spectroscopy and cortical microdialysis to observe intra- plus extracellular brain glucose variations. This method allows us to understand neurotransmitter systems underlying normal physiological function and behaviour. Because of the growing interest in exercise and brain functioning, it should be possible to investigate increasingly subtle behavioural and physiological changes within the central nervous system. There is now compelling evidence that regular physical activity is associated with significant physiological, psychological and social benefits in the general population. In contrast with our knowledge about the peripheral adaptations to exercise, studies relating exercise to brain neurotransmitter levels are scarce. It is of interest to examine the effect of short and long term exercise on neurotransmitter release, since movement initiation and control of locomotion have been shown to be related to striatal neurotransmitter function, and one of the possible therapeutic modalities in movement, and mental disorders is exercise therapy. Until very recently most experimental studies on brain chemistry were conducted with postmortem tissue. However, in part because of shortcomings with postmortem methods, and in part because of the desire to be able to directly relate neurochemistry to behaviour, there has been considerable interest in the development of 'in vivo' neurochemical methods. Because total tissue levels may easily mask small but important neurochemical changes related to activity, it is important to sample directly in the extracellular compartment of nervous tissue in living animals. Since the chemical interplay between cells occurs in the extracellular fluid, there was a need to access this compartment in the intact brain of living and freely moving animals. Estimation of the transmitter content in this compartment is believed to be directly related to the concentration at the site where these compounds are functionally released: in the synaptic cleft. As measurements in the synapse are not yet possible, in vivo measurements in the extracellular fluid appear to provide the most directly relevant information currently available. This article provides an overview of the in vivo microdialysis technique as a method for measuring in the extracellular space, and its application in exercise science. Although this technique has been used in different tissues such as brain, adipose tissue, spinal cord and muscle, in animals as well as humans, we will focus on the use of this in vivo method in brain tissue. Recently two excellent reviews on the application of microdialysis in human experiments especially in subcutaneous tissue have been published, and we refer the interested reader to these articles.