Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peak of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide.