The HNF-3 α, β and γ genes constitute a family of transcription factors that are required for hepatocytespecific gene expression of a number of genes, e.g. transthyretin, α-1 antitrypsin and tyrosine aminotransferase. These genes share a highly conserved DNA-binding domain first found in the Drosophila gene, forkhead, which is required for the normal patterning of the developing gut and central nervous system in Drosophila. In adult mouse tissues, transcripts from HNF-3 αand β have been localised to the liver, intestine and lung, whereas HNF-3 γ is found in the liver, intestine and testis. In light of the early developmental significance of forkhead in Drosophila, we have compared the patterns of expression of HNF-3 α, β and γ mRNAs during murine embryogenesis. We find that these genes are sequentially activated during development in the definitive endoderm. HNF-3 β mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated. Subsequently, HNF-3 α is transcribed in the primitive endoderm in the region of the invaginating foregut and HNF-3 γ appears upon hindgut differentiation. These genes have different anterior boundaries of mRNA expression in the developing endoderm and transcripts are found in all endoderm-derived structures that differentiate posterior to this boundary. Therefore, we propose that these genes define regionalisation within the definitive endoderm. Furthermore, differential mRNA expression of HNF-3 α and β is detected in cells of the ventral neural epithelium, chordamesoderm and notochord. In the neural epithelium, expression of HNF-3 αand β mRNA becomes localised to cells of the floor plate. We propose that, in addition to their characterised requirement for liver-specific gene expression, HNF-3 αand γ are required for mesoderm and neural axis formation. We also conclude that HNF-3 β is the true orthologue of the Drosophila forkhead gene.