The fork head/HNF-3 genes form a subclass of a family of transcription factors united by the possession of a conserved DNA binding domain known as the fork head domain. Most vertebrate HNF-3 class genes show several conserved sites of expression during development, including the dorsal lip/Hensen's node, notochord and floor plate, all structures known to organise adjacent tissues. In this paper I report the characterisation of HNF-3 class genes from the cephalochordate amphioxus. I show that amphioxus has two HNF-3 class genes, named AmHNF-3-1 and AmHNF-3-2; molecular phylogenetic analysis reveals that these derive from an independent duplication in the cephalochordate lineage. The expression of both genes in early development appears identical and shows striking similarities to that of vertebrates. In neurulae, transcripts of both genes were detected in the presumed organiser, endoderm, and notochord, supporting morphological and embryological evidence that these are homologous between vertebrates and amphioxus. This expression pattern overlaps considerably with that of amphioxus brachyury, suggesting that the functional relationship between these genes in vertebrates is conserved with amphioxus. Expression of both genes was maintained in the endoderm and notochord up to the 6 somite stage. After the 6 somite stage no expression of AmHNF-3-2 was detected and expression of AmHNF-3-1 began to decrease in the notochord, such that by the 10 somite stage transcripts were only detected in the terminal regions. At this stage, however, a column of AmHNF-3-1-expressing cells was detected at the ventral midline of the neural tube, a position occupied by the floor plate in vertebrates. This is the first evidence that amphioxus has a floor plate which is specified by a mechanism conserved with vertebrates. Taken together these data support two conclusions: Firstly, that the role of the dorsal lip/Hensen's node, notochord, and floor plate as organisers of the vertebrate body plan evolved prior to the separation of the vertebrate and cephalochordate lineages, at least 520 million years ago. Secondly, that the role of HNF-3 genes in these structures predates the origin of the multiple HNF-3 genes found in vertebrates.