Astrocytes are an abundant glial cell type of the central nervous system that appear to play a role in regulating extracellular potassium concentrations in brain, thereby contributing to the maintenance of normal neuronal activity. Voltage-gated potassium conductances, shown to be present in astrocytes, may be involved in this and other astrocytic functions. Toward defining the role of voltage-gated potassium channels in astrocytes, total RNA prepared from cultured mouse cortical astrocytes was screened, using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, for the expression of several members of the Shaker-like potassium channel subfamily (Kv1.1-Kv1.6). A relatively high level of Kv1.6 transcript was identified by RT-PCR and then confirmed and quantitated by ribonuclease protection assays using a Kv1.6-specific riboprobe. Immunocytochemical staining showed double-labeling of glial fibrillary acidic protein-positive cells with antibody specific for the Kv1.6 channel. The Kv1.6 protein expression was variable among the individual astrocytes. Outward voltage-gated currents were studied in astrocytes in primary culture using the Nystatin-perforated patch voltage clamp technique. Outward potassium currents were observed in all cells studied, and this current was partially blocked by perfusion with 100 nM dendrotoxin (DTX) in 14 of 16 cells tested. This DTX-sensitive current appeared to be a sustained outward potassium current, consistent with the suggestion that the Shaker-like potassium channel Kv1.6 underlies a portion of the delayed rectifier potassium current in cultured mouse cortical astrocytes.