We have characterized the structure of the chicken corticotropin-releasing factor (CRF) gene through cDNA cloning and genomic sequence analysis, and we analyzed the expression of CRF mRNA and peptide in the diencephalon of the chick throughout embryonic development. The structure of the chicken CRF gene is similar to other vertebrate CRF genes and contains two exons and a single intron. The primary structure of the mature chicken CRF peptide is identical to human and rat CRF. This is the first archosaurian CRF gene to be characterized. We used RIAs to analyze CRF peptide content in the diencephalon and the median eminence (ME), and plasma corticosterone during the last week of embryonic development. We also developed a semi-quantitative RT-PCR method to analyze the expression of CRF mRNA during the same period. CRF peptide content in the diencephalon increased, while peptide content in the ME decreased just before hatching, suggesting that release and biosynthesis are coupled. Plasma corticosterone concentration significantly increased between embryonic day 20 and the first day post-hatch. By contrast, CRF mRNA levels in the diencephalon decreased just before hatching. Changes in CRF production just before hatching may be causally related to the regulation of the thyroid and interrenal axes at this stage of chicken development.