Hyperpolarization-activated currents were recorded from rat brain cortical and spinal cord astrocytes maintained in culture. Spinal cord astrocytes expressed primarily an inward rectifier potassium current characterized by time-dependent inactivation, a strong dependence on extracellular Na+ and insensitivity to intracellular GTP-gamma-S (0.2 mM). In cortical astrocytes voltage clamp protocols aimed to elicit currents activated at, or negative to cell membrane potentials led to the development of two distinct ion currents. The most prominent current resembled the inward rectifier potassium current. This component was sensitive to blockade by extracellular cesium and was greatly reduced during recordings performed with GTP-gamma-S (0.2 Mm) added to the pipette solutions. The remaining current component was similar to the endothelial I ha current. I ha conductance was enhanced by extracellular potassium and the current reversal potential behaved as expected for a mixed cation, Na+/K+ current. I ha was nearly abolished after removal of extracellular Na. These results are consistent with the expression of a novel mixed cation conductance in glial cells, possibly involved in extracellular potassium buffering.