Glutamate microsensors form a promising analytical tool for monitoring neuronally derived glutamate directly in the brain. However, when a microsensor is implanted in brain tissue, many factors can diminish its performance. Consequently, a thorough characterization and evaluation of a microsensor is required concerning all factors that may possibly be encountered in vivo. The present report deals with the validation of a hydrogel-coated glutamate microsensor. This microsensor is constructed by coating a carbon fiber electrode (10-microm diameter; 300-500 microm long) with a five-component redox hydrogel, in which L-glutamate oxidase, horseradish peroxidase, and ascorbate oxidase are wired via poly(ethylene glycol) diglycidyl ether to an osmium-containing redox polymer. A thin Nafion coating completes the construction. Although this microsensor was previously used in vivo, information concerning its validation is limited. In the present study, attention was given to its selectivity, specificity, calibration, oxygen dependency, biofouling, operating potential dependency, and linear range. In addition, successful microsensor experiments in microdialysate, in vitro (in organotypic hippocampal slice cultures), and in vivo (in anesthesized rats) are shown.