Antisera recognizing dynorphin 1-13, but not other opioid peptides with a high avidity, have been generated in rabbits. Using a radioimmunoassay technique levels of dynorphin-immunoreactivity (dyn-ir) have been measured in the brain and pituitary of rats. The concentrations of dyn-ir ranged from a maximum in the intermediate/posterior lobe of the pituitary (about 1200 pmol/g) to a minimum in the cerebellum (about 1 pmol/g) as follows: pituitary intermediate/posterior lobe greater than adenohypophysis greater than hypothalamus greater than hippocampus = striatum = midbrain = thalamus = medulla/pons greater than cortex greater than cerebellum. Gel-filtration of hypothalamic extracts revealed 4 immunoreactive components with apparent molecular weights (MW) of 3500, 2400, 1300 and less than 1000 daltons, respectively. No dyn-ir was found to elute as dynorphin1-13 (MW = 1700). The 2400 and 1300 dalton materials showed opiate-like activity on the guinea-pig ileum longitudinal muscle preparation, indicating that a substantial part of the dyn-ir measured represented biologically active material.