1. The authors investigated the effect of the benzodiazepine receptor antagonist flumazenil on freezing behavior induced by conditioned fear stress and used a time-sampling procedure. 2. Rats were individually subjected to 5 min of inescapable electric footshock in a shock chamber. Twenty-four hours after the footshock, the rats were again placed in the shock chamber and observed for 5 min without shocks: this procedure is termed conditioned fear stress. 3. Subcutaneous administration of flumazenil (10 mg/kg) 30 min before conditioned fear stress reduced conditioned freezing, while flumazenil (1-10 mg/kg) 30 min before footshock did not. This indicates that flumazenil reduced the expression of conditioned fear, suggesting an anxiolytic effect of flumazenil. 4. This effect may be attributable to antagonism by flumazenil to endogenous inverse agonist-like ligands for benzodiazepine receptors. It is suggested that endogenous inverse agonist-like ligands for benzodiazepine receptors are involved in the expression of conditioned fear stress.