Corticotropin-releasing-factor-like immunoreactivity (CRF-LI) was measured in a number of subcellular fractions from rat brain using a highly sensitive and specific radioimmunoassay. CRF-LI was highly enriched in the crude synaptosomal/ mitochondrial fraction (P<sub>2</sub>) relative to the homogenate, P<sub>1</sub>, S<sub>1</sub>, and S<sub>2</sub> fractions. Separation of the P<sub>2</sub> fraction into synaptosomal, myelin, and mitochondria-enriched subfractions on a rapid one-step sucrose gradient revealed that CRF-LI was present at higher concentrations in the synaptosomal fraction than in the mitochondrial and myelin fractions. The distribution of CRF-LI paralleled that of synapsin, a synaptic vesicle marker phosphoprotein, but not that of pyruvate dehydrogenase, a mitochondrial phosphoprotein. These results are consistent with a nerve terminal localization of CRF and a potential role for this peptide as a central nervous system neurotransmitter.