Primary cultures of mouse cortical astrocytes were used to investigate the changes in intracellular Na+ concentration ([Na+]i) following exposure to the excitatory neurotransmitter glutamate. The fluorescent probe sodium-binding benzofuran isophthalate (SBFI) was used to measure [Na+]i by epifluorescence microscopy imaging. Detection of the low light level fluorescent signal was accomplished using a Gen III+ intensified CCD camera. Fluorescence excitation ratio images of dye-loaded cells were obtained after sequential illumination at 340nnm and 380nm, with an emission observed at >520nm. Ratio images were proportional in intensity to [Na+]i. In situ calibration of the fluorescent signals was obtained for each experiment and each cell under study by equilibrating [Na+]i with external Na+ after treatment with the cation ionophores monensin and gramicidin, and with ouabain, a specific inhibitor of the Na+/K+ ATPase. Cells on glass coverslips were perfused at 35°C in a closed microscope chamber using solutions buffered with 25 mM bicarbonate/5%CO2.