In addition to its endocrine action in the anterior pituitary, corticotropin-releasing factor (CRF) also appears to play a role in regulating higher central nervous system function(s). To investigate further the role of CRF in brain, a specific RIA was used to measure in vitro CRF release during incubation of various rat brain regions in Krebs-Henseleit buffer. Increasing the potassium concentration to 56 mM resulted in a 10-fold increase in CRF release from minced hypothalamus. However, the high K+ concentrations had no effect in the presence of calcium-free buffer containing 1 mM EGTA. Scorpion venom also stimulated CRF release in a calcium-dependent manner. CRF-like immunoreactivity was detected in extrahypothalamic brain regions using RIA and HPLC. Significant calcium-dependent CRF release from the rat amygdala and midbrain was observed in response to 56 mM K+ or scorpion venom. These results indicate that depolarizing agents induce the release of CRF-like immunoreactivity not only from the hypothalamus but also from other rat brain regions and lend further support to the hypothesis that CRF may be a neurotransmitter or neuromodulator in the central nervous system.