Effects of 20-hydroxyecdysone (20-HE), a neurosteroid, on cultured rat cortical neurons were examined using the whole cell recording technique. Under the voltage and current clamp conditions, brief application (5 sec) of 20-HE alone did not produce current changes nor any changes in the membrane potential. However, the chemical dose-dependently potentiated the GABA-induced current and hyperpolarization, which were blocked by bicuculline. These results suggest that 20-HE acts on the modulatory site of the GABAA receptor and potentiates GABAergic inhibition in rat cortical neurons.