Local pharmacological manipulations of both pupils in persons with cluster headache (CH) have shown a reduced pain-side sympathetic activity. It is difficult to determine if this sympathetic defect is localized in the nuclei of the CNS and/or in peripheral neurons that innervate the pupil. This study demonstrates that in a CH group 2% tyramine (an intraneuronal norepinephrine releaser) instillation into both eyes induces an asymmetric and bilateral mydriasis with the onset of anisocoria characterized by a pupillary diameter being less on the pain-side eye. In addition, intravenous administration of 0.10 mg clonidine, an inhibitor of central sympathetic activity, causes a bilateral miotic response, which is more marked on the pain-side eye. In a healthy control group, clonidine induces a symmetric and bilateral miosis but less intense than that observed in both eyes of CH sufferers. In CH patients, pretreatment with clonidine augments the degree of anisocoria induced by tyramine instillation, increasing the mydriatic response only in the pain-free-side pupil. The hypothesis of a permanent sympathetic defect of the pain-side pupil expressing itself as a reduced sympathetic tone of CNS nuclei and peripheral neurons that innervate the pupil is proposed.