In spite of several studies showing specific physiological functions of changes in the extracellular level of the major excitatory and inhibitory transmitters, Glu and GABA within the brain ([Glu]EXT, [GABA]EXT) the exact origin (neuronal vs. astroglial, synaptic vs. extrasynaptic) of Glu and GABA present in dialysate samples is still a matter of debate. For better understanding the significance of in vivo microdialysis data, here we discuss methodological details and problems in addition to regulation of [Glu]EXT and [GABA]EXT. Changes in [Glu]EXT and [GABA]EXT under pathological conditions such as ischemia and epilepsy are also reviewed. Based on recent in vivo microdialysis data we argue that ambient [Glu] EXT and [GABA]EXT may have a functional role. It is suggested that specific changes in concentrations of Glu and GABA in dialysate samples together with their alterations independent of neuronal activity indicate the involvement of Glu and GABA in the information processing of the brain as essential signaling molecules of nonsynaptic transmission as well. Since various drugs are able to interfere with extrasynaptic signals in vivo, studying the extracellular cell-to-cell communication of brain cells represents a new aspect to improve drugs modulating Gluergic as well as GABAergic neurotransmission.