Valerian extracts have been used for centuries to alleviate restlessness and anxiety albeit with unknown mechanism of action in vivo. We now describe a specific binding site on GABA(A) receptors with nM affinity for valerenic acid and valerenol, common constituents of valerian. Both agents enhanced the response to GABA at multiple types of recombinant GABA(A) receptors. A point mutation in the beta2 or beta3 subunit (N265M) of recombinant receptors strongly reduced the drug response. In vivo, valerenic acid and valerenol exerted anxiolytic activity with high potencies in the elevated plus maze and the light/dark choice test in wild type mice. In beta3 (N265M) point-mutated mice the anxiolytic activity of valerenic acid was absent. Thus, neurons expressing beta3 containing GABA(A) receptors are a major cellular substrate for the anxiolytic action of valerian extracts.