Valerian is commonly used for the treatment of insomnia and anxiety. Valerian extracts allosterically modulate GABA-A receptors and induced an anxiolytic activity. This activity is closely related to valerenic acid. In the present experiments it was investigated whether acetoxy valerenic acid may interfere with the anxiolytic action of valerenic acid. Situational anxiety was measured using male CD-1 mice in the elevated plus maze test after oral administration of the test substances. In addition the body core temperature was measured. For the 3H-GABA binding assay dissected tissue from frontal cortex of male RjHan:WI rats were used. Statistical evaluation was performed by means of the non-parametric Kruskal-Wallies H-test, followed by the two-tailed Mann-Whitney U-test. Adding of acetoxy valerenic acid abolished the anxiolytic action of valerenic acid. There was no effect on body core temperature. Moreover, the valerian extract did not show any affinity to benzodiazepine binding sites. The determining compound for the observed anxiolytic effect of the valerian extract is its content of valerenic acid.