The distribution of preprodynorphin messenger RNA-containing perikarya in the central nervous system of the rat was determined with in situ hybridization histochemistry using a 35S-labelled complementary RNA probe. All of the regions of the central nervous system reported by other investigators to contain perikarya that synthesize prodynorphin-derived peptides, except the pedunculopontine tegmental nucleus, the accessory trigeminal nucleus, and the ventral nucleus of the trapezoid body, also contained perikarya that synthesize preprodynorphin messenger RNA. However, the olfactory bulb, the anterior olfactory nucleus, the islands of Calleja, the CA1-CA3 fields of the hippocampus, the septohippocampal nucleus, the diagonal band of Broca, the basal and cortical amygdaloid nuclei, the entopeduncular nucleus, the subthalamic nucleus, the superior colliculus, the Edinger-Westphal nucleus, the dentate nucleus, the raphes linearis and pontis, the dorsal cochlear nucleus, the medial vestibular nucleus, the inferior olive, and the dorsal motor nucleus of the vagus nerve also contained preprodynorphin messenger RNA-synthesizing perikarya. These observations suggest that prodynorphin-derived peptides have a much more pervasive role in central nervous system function than previously suspected. However, before the physiological significance of these observations can be judged, it will be necessary to determine whether all of the novel sites of preprodynorphin messenger RNA synthesis are sites of prohormone synthesis and conventional processing.