Abstract We have developed a highly sensitive and specific immunoassay for human/rat corticotrophin-releasing factor-41 (CRF-41) to enable determination of immunoreactive CRF-41 levels in biological samples. To achieve high specificity, sensitivity and speed we have used two antisera in a sandwich enzyme immunoassay combined with enzyme amplification. The assay has a sensitivity of 0.08 fmol/well compared with radioimmunoassay sensitivities of 0.5 fmol/tube and is highly specific for the intact CRF-41 molecule. Measurement of samples is complete within 24 h compared with the 5 days required to obtain sensitive radioimmunoassay measurement. The assay has been used to measure both rat hypothalamic CRF-41 tissue content and release in vitro with good correlation when compared to radioimmunoassay measurement using antisera rC70 (0.983) or R1 (0.953). The assay only measures immunoreactive CRF-41 coeluting with human/rat CRF-41 and its oxidized form Met [O(21,38)]CRF-41 in human and rat tissue extracts separated by high-performance liquid chromatography. The ability to measure immunoreactive CRF-41 in unextracted plasma allows rapid measurement and eliminates multiple extraction steps.