Capillary zone electrophoresis is capable of analyzing nanoliter volumes, reducing the challenge posed by brain microdialysis time resolution improvement to the management of nanoliter dialysate volumes. This fact has not been overlooked and 12- and 6-s time resolution microdialysis have been reported in anesthetized rats. However, behavioral experiments require fully awake and freely moving animals. To achieve high temporal resolution brain microdialysis in awake unrestrained rats, we have developed an online device that mixes the outflowing dialysate with fluorescein isothiocyanate and buffer within a 26-nl reactor. The mixture was continuously accumulated in a 99-micrometer-bore capillary tube. After the experiment the tube was cut into 4-mm pieces and the content of each piece (30 nl, equivalent to 1 s dialysate) was transferred to a test tube. After allowing 18 h for derivatization, the samples were diluted with water and injected into a capillary electrophoresis laser-induced fluorescence detection instrument. This protocol was tested first in an in vitro assay and proved to be capable of detecting glutamate concentration changes in only 1 s. For the in vivo assays, a probe was inserted into the primary somatosensory cortex of eight rats divided in two groups. One group was stimulated by gently moving its whiskers for 10 s. The other group had no whisker manipulation. Moving the whiskers released glutamate in the experimental group. The first and only change was observed at the 12th s. This method allows 1-s time resolution brain microdialysis in freely moving rats and multiple amino acid analysis every second during sensory perception or motor actions in behavioral experiments.