Microdialysis was coupled on-line with derivatization by o-phthalaldehyde and β-mercaptoethanol and optically gated capillaiy electrophoresis to determine D- and L- aspartate in tissue samples obtained from rats. The microdialysis probe was inserted into a homogenized tissue sample which allowed generation of a continuous sample stream that was filtered and deproteinated. With 7.5 mM β-cyclodextrin (CD) in the electrophoresis buffer, the enantiomers of interest could be resolved in 3 s with an electric field of 2500 V/cm over a separation length of 15 mm. Values of D- and L-aspartate in different tissues agreed well with those obtained by an HPLC procedure that required protein precipitation, centrifugation, and extraction. The speed and compatibility with automation of the microdialysis/CE method may make it a general approach for a variety of applications involving high-throughput analysis or sensorlike operation.