A specific antiserum was prepared against dynorphin B, an endogenous opioid peptide contained in a recently isolated 4,000-dalton dynorphin. The antiserum did not crossreact with dynorphin A, alpha-neo-endorphin, beta-neo-endorphin, dynorphin-(1-8), or [Leu]enkephalin. In immunohistochemical staining experiments on frozen sections through rat brains from normal and colchicine-treated animals, the antiserum labeled the same neuronal fiber systems previously described as containing both dynorphin A and alpha-neo-endorphin immunoreactive material. The alpha-neo-endorphin/dynorphin A immunoreactive perikarya in the hypothalamic magnocellular nuclei also were labeled by the dynorphin B antiserum. In addition, the dynorphin B antiserum revealed groups of immunoreactive neuronal cell bodies in several other hypothalamic and extrahypothalamic areas, including brain-stem, midbrain, central nucleus of amygdala, and in the dorsomedial, lateral, and anterior nuclei of hypothalamus. These perikarya had not been detected in previous studies that used dynorphin A and alpha-neo-endorphin antisera. The findings are in agreement with recent studies demonstrating a common precursor for dynorphin A, dynorphin B, and alpha-neo-endorphin. The apparently wider distribution of dynorphin B immunoreactive cell bodies compared to alpha-neo-endorphin/dynorphin A immunoreactive perikarya may be a reflection of differential processing of the precursor in different brain regions.