Stepwise processing of the prohormone for dynorphin-related peptides, proenkephalin B, may generate a large number of opioid peptides. It is therefore important to perform a chemical characterization of the immunoreactive dynorphin (ir-dyn) found in different tissues. In this study dynorphin peptides in human substantia nigra were characterized. Highly sensitive radioimmunoassays (RIA) directed against the C-terminals of dynorphin A (dyn A) and dynorphin B (dyn B) respectively, were used to measure the levels of dynorphin peptides. Tissue extraction was performed either with MeOH/0.1 M HCl (1/1) or 1 M HAc. Gel filtration on a Sephadex G-50 column revealed three peaks of ir-dyn B, the predominating one coeluting with synthetic dyn B. Ir-dyn A also appeared in three peaks, one of them (20-30%) coeluting with dyn A. The HAc extract contained much higher levels of ir-dyn B as compared to the MeOH/HCl extract, but approximately the same levels of ir-dyn A. Ion exchange separation of the HAc extract somewhat changed the size distribution pattern, with more ir coeluting with the synthetic peptides compared to other molecular weight forms.