The extracellular concentration of potassium ([K+]o) in brain tissue is modified by neuronal activity and is increased under several pathological conditions. The influence of neurosteroids on the astroglia response to high [K+]o was assessed on cultured slices from rat hippocampus. Exposure to [K+]o above physiological (3 mM) levels resulted in the progressive appearance of cell processes immunoreactive for glial fibrillary acidic protein (GFAP). The maximal effect was observed at 50 mM [K+]o, and further increases of [K+]o did not increase the extension of GFAP-immunoreactive processes. The effect was observed as early as 10 min after increasing [K+]o, was independent of new protein synthesis, and was reversible, reaching control conditions by 15 h after resetting [K+]o to physiological levels. Gonadal hormones and neurosteroids had prominent and variable effects on the stimulatory influence of high [K+]o on astroglia morphology. At physiological [K+]o, 17beta-estradiol and pregnenolone, as well as its sulfate derivative, increased the extension of GFAP-immunoreactive processes. However, at high [K+]o, testosterone, pregnenolone, and dehydroepiandrosterone and its sulfate derivative decreased the extension of GFAP-immunoreactive processes. Effects of gonadal hormones and neurosteroids were blocked by the protein synthesis inhibitor cycloheximide. These results suggest that non-genomic effects of high [K+]o on glial cells interact with genomic effects of steroids to modulate astroglia morphology.