This study demonstrates a fluorescence method for in vivo sensing of the dynamic change of Zn(2+) concentration in auditory cortex microdialysates induced by salicylate with N'-(7-nitro-2,1,3-benzoxadiazole-4-yl)-N,N,N'-tris(pyridine-2-ylmethyl) ethane-1,2-diamine (NBD-TPEA) as a probe. The excellent properties of the NBD-TPEA probe make it possible to achieve a high selectivity for Zn(2+) sensing with the co-existence of amino acids and other metal ions as well as the species commonly existing in the cerebral system. To validate the method for in vivo fluorescence sensing of Zn(2+) in the rat brain, we pre-mix the microdialysates in vivo sampled from the auditory cortex with the NBD-TPEA probe and then perfuse the mixtures into a fluorescent cuvette for continuous-flow fluorescence detection. The method demonstrated here shows a linear relationship between the signal output and Zn(2+) concentration within the concentration range from 0.5 μM to 4 μM, with a detection limit of 156 nM (S/N = 3). The basal level of extracellular Zn(2+) in auditory cortex microdialysates is determined to be 0.52 ± 0.082 μM (n = 4). This value is increased by the injection of 100 mg mL(-1) of salicylate (1 μL min(-1), 5 min, i.p.), reaches a peak at the time point of 90 min, and levels off with time. Such an increase is attenuated by the injection of MK-801, a potent and specific NMDA receptor antagonist, after the pre-injection of 100 mg mL(-1) salicylate for 5 min. This study offers a fluorescence method for in vivo sensing of Zn(2+) in the rat brain that could be useful for the investigations of chemical processes involved in brain functions.