The effects of inhibitors of the glial Na+/glutamate co-transporter on the intracellular Na+ concentration (Na+) were investigated in mouse cortical astrocytes. Na+ was monitored by fluorescence microscopy on single astrocytes using the Na+-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-beta-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in robust and reversible increases in Na+ that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the Na+ response to these compounds was concentration-dependent with EC(50) values of 11.1 microM (THA) and 7.6 microM (t-PDC), as was the initial rate of Na+ rise (EC(50) values of 14.8 microM for THA and 11.5 microM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-beta-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of Na+ up to a concentration of 500 microM . TBOA inhibited the Na+ response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) values of 114 and 63 microM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked Na+ increase by TBOA was approximately 70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters.