The author examined the acute anxiolytic effects of monoamine oxidase inhibitors on freezing behavior, a putative index of anxiety, induced by conditioned fear stress. The selective serotonin1A receptor agonist inhibited freezing dose dependently. The irreversible, non-selective monoamine oxidase inhibitors tranylcypromine (3 and 15 mg/kg) and phenelzine (30 and 80 mg/kg) reduced freezing significantly. Clorgyline (10 mg/kg, irreversible selective monoamine oxidase A inhibitor), Ro 41-1049 (30 mg/kg, reversible selective monoamine oxidase A inhibitor), selegiline (3 mg/kg, irreversible selective monoamine oxidase B inhibitor) and lazabemide (10 mg/kg, reversible selective monoamine oxidase B inhibitor) had no effect on freezing behavior. However, combined administration of clorgyline (10 mg/kg) and selegiline (3 mg/kg) reduced freezing significantly, as well as combined administration of clorgyline (10 mg/kg) and lazabemide (10 mg/kg), Ro 41-1049 (30 mg/kg) and selegiline (3 mg/kg), or Ro 41-1049 (30 mg/kg) and lazabemide (10 mg/kg). These effects of monoamine oxidase inhibitors on freezing were not due to non-specific motor effects. These results suggest that acute inhibition of both monoamine oxidase A and B reduces anxiety or fear, while inhibition of monoamine oxidase A or B alone fails to reduce anxiety or fear. In vivo microdialysis studies showed that the irreversible monoamine oxidase A inhibitor clorgyline and the irreversible monoamine oxidase B inhibitor selegiline induced a mild increase and no increase in extracellular serotonin, respectively. Interestingly, the combined treatment with clorgyline and selegiline resulted in much larger increases in extracellular serotonin in the medial prefrontal cortex than did either monoamine oxidase inhibitor alone. Our previous studies have indicated that facilitation of 5-HT neurotransmission decreases conditioned freezing, i.e., anxiety or fear. The results of these in vivo microdialysis studies may account for the results of this study that the simultaneous blockade of both monoamine oxidase A and B reduced conditioned freezing, whereas blockade of either monoamine oxidase alone failed.