alpha-Neo-endorphin and dynorphin immunoreactivities in rat brain were visualized by double antibody immunofluorescence of frozen sections. Antibodies were used that were specific for their respective antigens. The pattern of neuronal immunostaining produced by alpha-neo-endorphin and dynorphin antisera in adjacent serial sections was completely superimposible. No areas were found in which alpha-neo-endorphin or dynorphin immunoreactivities existed alone. The following brain regions contained alpha-neo-endorphin/dynorphin-immunoreactive fibers and terminals: the median forebrain bundle, the internal capsule, the substantia nigra, the hypothalamus, the nucleus accumbens, the hippocampus, and the medulla oblongata. A few fibers were seen in the cerebral cortex and in the corpus striatum. In many regions, this neuronal fiber system seems to overlap the neuronal system previously described to contain [Met]-/[Leu]enkephalin-immunoreactive material. In brains from colchicine-treated animals, numerous alpha-neo-endorphin/dynorphin-immunoreactive neuronal cell bodies were seen in the supraoptic, retrochiasmatic supraoptic, paraventricular, and magnocellular accessory nuclei of the hypothalamus. It is concluded that alpha-neo-endorphin-like and dynorphin-like immunoreactivities are part of the same neuronal system.