A cobalt-glucose-oxidase diaminobenzidine (Co-GOD) method, employing a specific antiserum against rat corticotropin releasing factor (CRF), was applied to determine immunohistochemically a widespread and detailed localization of corticotropin releasing factor-like immunoreactivity (CRFI) in the rat brain. Besides the CRFI cells in the paraventricular hypothalamic nucleus that project to the median eminence, CRFI cells were demonstrated in many brain regions, including the olfactory bulb, cerebral cortex, septal nuclei, hippocampus, amygdala, thalamic nuclei, medial hypothalamic nuclei, lateral hypothalamic area, perifornical area, central gray, cuneiform nucleus, inferior colliculus, raphe nuclei, mesencephalic reticular formation, laterodorsal tegmental nucleus, locus coeruleus, parabrachial nuclei, mesencephalic tract of the trigeminal nerve, pontine reticular formation, lateral superior olive, vestibular nuclei, prepositus hypoglossal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, lateral reticular nucleus, nucleus of the spinal tract of the trigeminal nerve, external cuneate nucleus, inferior olive, and medullary reticular formation. CRFI-reacting neural processes were also detected in these same areas. In particular, the median eminence, lateral septum, bed nucleus of the stria terminalis, mesencephalic reticular formation, parabrachial nuclei, and nucleus of the solitary tract contained large numbers of CRFI fibres. The widespread localization of CRFI demonstrated in the present study strongly suggests that CRF, like many other neurohormones and peptides, may act as a neurotransmitter and/or neuromodulator in numerous extrahypothalamic circuits, as well as participate in neuroendocrine regulation.