We report here that corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in the rat central nervous system (CNS). In frontoparietal cortex homogenates, the stimulation by CRF was dependent on time, temperature, tissue protein concentration, and guanine nucleotides. The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity [(Nle21,38) rat CRF greater than rat CRF approximately equal to acetyl ovine CRF (4-41) approximately equal to alpha helical ovine CRF greater than ovine CRF much greater than ovine CRF (1-39) approximately equal to ovine CRF (7-41)] was consistent with their affinities for CRF receptors in the brain and their relative potencies in stimulating pituitary adrenocorticotropic hormone secretion in vitro. The putative CRF receptor antagonist, alpha helical ovine CRF (9-41), did not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) production but was able to attenuate the stimulation by various concentrations of rat CRF. The regional distribution of 125I-Tyr(o)-ovine CRF binding (olfactory bulb greater than frontoparietal cortex approximately equal to cerebellum greater than hypothalamus greater than striatum greater than or equal to midbrain greater than hippocampus greater than or equal to spinal cord) did not correspond with the regional degree of CRF receptor-mediated stimulation of adenylate cyclase (frontoparietal cortex greater than olfactory bulb greater than or equal to cerebellum greater than midbrain greater than or equal to hippocampus greater than striatum greater than or equal to hypothalamus greater than spinal cord). In addition, marked differences were observed in the ability of forskolin to potentiate CRF-stimulated cAMP production in the various brain areas examined. In summary, these data demonstrate that at least one of the second-messenger systems mediating the effects of CRF in the CNS involves stimulation of cAMP production and provides further support for a neurotransmitter role for this neuropeptide in the brain. Significant differences in the regulation of CRF-stimulated cAMP production and the disparity between CRF receptor number and receptor-mediated adenylate cyclase activity in discrete regions of the rat CNS suggest that some populations of CRF receptors in the brain may be functionally coupled to alternative signal transduction mechanisms.