Astrocytes have a function in the uptake of excitatory neurotransmitter L-glutamate via the glutamate transporter. To evaluate the L-glutamate clearance capacity of atrocytes, we developed a colorimetric method for the determination of L-glutamate concentration and measured changes in extracellular L-glutamate concentration in rat cortical astrocyte cultures. When L-glutamate (50-200 μM) was added to astrocyte cultures and incubated for 1-8 h, the extracellular L-glutamate concentration declined with time. When L-glutamate was mixed with astrocyte culture supernatants only, no significant change in L-glutamate concentration was observed, ruling out the possibility that L-glutamate is spontaneously or enzymatically degraded in the extracellular space. Alternatively, the time-dependent decline of extracellular L-glutamate concentration was blocked by the presence of glutamate uptake inhibitors, indicating that the glutamate uptake system of astrocytes plays a major role in the clearance of extracellular L-glutamate.