Immunoreactive β-endorphin was measured in the ventricular fluid of six patients with chronic pain. Stimulation of the periaqueductal gray matter in three patients with pain of peripheral origin resulted in significant increases (50 to 300 percent) in the concentration of ventricular immunoreactive β-endorphin. In three other patients suffering deafferentation dysesthesia, stimulation of the posterior limb of the internal capsule did not alter the concentration of this peptide. These results provide evidence of the release of human immunoreactive β-endorphin in vivo and suggest that naloxone-reversible pain relief achieved by stimulation of the periaqueductal gray matter may be in part mediated by the activation of β-endorphin-rich diencephalic areas.