The distribution of immunoreactive dynorphin (ir-dyn) has been determined in various regions of human brain and pituitary by use of a highly specific radioimmunoassay. The concentrations of ir-dyn in the substantia nigra (24.5 pmol/g) and hypothalamus were among the highest in the 26 brain areas examined. Substantial amounts were also measurable in other extrapyramidal structures such as the caudate nucleus, pallidus and putamen. Lower concentrations of ir-dyn were detected in the amygdala, hippocampus, periaqueductal gray matter, colliculi, pons, medulla and area postrema, but only low amounts were found in the posterior lobe of the pituitary, while no ir-dyn was detectable in the anterior lobe. By gel permeation chromatography the brain immunoreactivity was shown to consist of 3-4 peaks of apparent molecular weights of about 12,000, 6000, 1800 and less than 1000. It was possible to demonstrate the high opioid potency of 2 of these peaks in the guinea-pig ileum longitudinal muscle bioassay after purification by immunoprecipitation. A comparison of the distribution pattern of ir-dyn revealed some parallels with enkephalin, whereas the distribution of ir-beta-endorphin is quite different.