In the pyramidal cell layer of the CA1 region of the hippocampus in the urethane-anesthetized adult rat, there is an initial alkalinization followed by an acidification in response to synchronized seizure activity induced by stimulus trains. In this study, the role of astrocytes in these extracellular pH changes during neuronal activity was examined using local injection of two relatively selective glial toxins (fluorocitrate (FC) and fluoroacetate (FA)) into the CA1 cell layer. Both glial toxins reduced the peak level of acidification reached after 20 Hz stimulus trains to the contralateral CA3 region, without changing the lengthening of the afterdischarge, when compared to animals that had received a local injection of vehicle. After administration of either glial toxin, the peak level of acidification still correlated with the total discharge duration, but the levels of acidification were consistently lower than in control animals. Administration of either glial toxin had no effect on the peak alkalinization during the stimulus train, or on the rate of recovery from peak level of acidification. Injection of either vehicle, FA, or FC had no effect on the amplitude or frequency of the neuronal discharge during the afterdischarge. The results suggest that, in normal conditions, astrocytes contribute to the acidification of the extracellular space that occurs in response to intense neuronal activity. This acidification may contribute to feedback regulation of neuronal excitability.