The sensitivity of the recombinant alpha 6 beta 2 gamma 2 GABAA receptor expressed in HEK 293 cells to neuroactive steroids was studied. The steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), pregnenolone sulfate and 3 alpha-OH-DHP sulfate have different modulatory effects on [3H]muscimol or [3H]Ro15-4513 binding to the alpha 6 beta 2 gamma 2 than to the alpha 1 beta 2 gamma 2 receptor. Binding of both radioactive ligands to the alpha 6 beta 2 gamma 2 receptor was maximally potentiated with each steroid used (10 nM) and decreased with further increases in steroid concentration. Using whole-cell recording, the GABA response of clusters of transfected HEK 293 cells was strongly potentiated by 3 or 10 nM 3 alpha-OH-DHP. In contrast, this response was reduced by 100 nM 3 alpha-OH-DHP. This latter effect appears to be related to the acceleration of the GABA response desensitization, observed in isolated cells. 3 alpha-OH-DHP (10 or 100 nM) was able to activate a response in the absence of GABA. It is proposed that the interaction of neuroactive steroids with the alpha 6 beta 2 gamma 2 receptor involves at least two distinct binding sites. One of them might be located close to the GABA binding site.