Accumulated evidence indicates that the core genetic mechanisms regulating early patterning of the brain rudiment in vertebrates are very similar to those operating during development of the anterior region of invertebrate embryos. However, the mechanisms underlying the morphological differences between the elaborate vertebrate brain and its simpler invertebrate counterpart remain poorly understood. Recently, we hypothesized that the emergence of the most anterior unit of the vertebrate brain, the telencephalon, could be related to the appearance in vertebrates' ancestors of a unique homeobox gene, Anf/Hesx1(further Anf), which is absent from all invertebrates and regulates the earliest steps of telencephalon development in vertebrates. However, the failure of Anf to be detected in one of the most basal extant vertebrate species, the lamprey, seriously compromises this hypothesis. Here, we report the cloning of Anf in three lamprey species and demonstrate that this gene is indeed expressed in embryos in the same pattern as in other vertebrates and executes the same functions by inhibiting the expression of the anterior general regulator Otx2 in favour of the telencephalic regulator FoxG1. These results are consistent with the hypothesis that the Anf homeobox gene may have been important in the evolution of the telencephalon.