To allow rat brain lactate measurement in vivo, a specific sensor based on a carbon fiber (phi = 30 microns) microelectrode coated with lactate oxidase was prepared. Combined with the differential normal pulse voltammetry measurement method, such a sensor, with a sensitivity of 9.15 +/- 0.91 mA.M-1.cm-2, provided a lactate linear response in concentrations ranging from 0.1 to 2.0 mM. The measurements performed appeared to be essentially insensitive to usual interference caused by the electroactive compounds present in the brain (ascorbic acid and peptides). In vivo detection performed in the cortex of the anesthetized rat led to the determination of a lactate concentration of 0.41 +/- 0.02 mM. Moreover, to validate the results obtained in vivo, an ex vivo determination of the lactate level was also performed in samples of brain tissue, plasma, and cerebrospinal fluid, using both voltammetry and a clinical analyzer with colorimetric-based detection. A good correlation was observed between the sets of data established by both methods.