Preembedding immunoperoxidase staining methods were used to permit ultrastructural analyses of the distribution in rat brain and pituitary of the corticotropin-releasing factor-binding protein (CRF-BP), a moiety distinct from CRF receptors, but which is nonetheless capable of binding the peptide and reversibly neutralizing its biological actions. In anterior pituitary, CRF-BP immunoreactivity (ir) was detected in corticotropelike cells, with reaction product associated principally with secondary lysosomes and multivesicular bodies and not at all with secretory granules. In brain, marked regional differences in the subcellular pattern of CRF-BP staining were evident. In isocortex, where BP/peptide colocalization is rare, BP-ir was distributed in cells and processes in a manner similar to that of a prototypic neuropeptide, including in terminals commonly engaging in synaptic contacts with unlabeled dendritic profiles. In the bed nucleus of the stria terminalis, a site that contains overlapping accumulations of CRF-BP-ir projections and CRF-ir perikarya, BP staining was restricted to vesicle-laden varicosities that rarely engaged in synaptic contacts with somatic or dendritic elements but were frequently apposed to unlabeled axon varicosities and terminals. In the ventromedial medulla, a site of partial CRF/BP overlap, most cells displayed a subcellular localization CRF-BP-ir like that seen in cortex, whereas in others the distribution shared similarities with that observed in pituitary. The results suggest that the function of the CRF-BP may differ in different cellular contexts. In cellular targets of CRF or in neurons in which peptide and BP coexist, the CRF-BP may play a role in processing and degradation of CRF and/or ligand-receptor complexes. In other areas of the central nervous system, the BP seems positioned to serve as a transmitter/modulator at conventional synapses or as an autocrine or paracrine modulator of local CRF effects.