Axial patterning in the nervous system of vertebrate embryos depends on inductive signals that derive from the organizer region (the dorsal lip in amphibians and the node in birds and mammals) and later from the notochord and floor plate. Previous studies have shown that Pintallavis, a member of the HNF-3/fork head transcription factor family, is expressed selectively by these cell groups in frog embryos and may be involved in regulating neural development. We report here that in early rat mouse embryos, the embryonic endoderm, the node, the notochord and the floor plate express two related transcription factors, HNF-3α and HNF-3β, which also function in the control of liver cell differentation. Early embryonic tissues express variant forms of HNF-3β which derive from the use of 5′ alternative exons. Within the organizer region and notochord, HNF-3β and HNF-3α have distinct temporal patterns of expression and appear in partially overlapping domains. The early expression pattern of mammalian HNF-3β in the node, notochord and midline neural plate cells is similar to that of Pintallavis in frog embryos. There does not appear to be a Pintallavis homologue in mice. This prompted us to isolate and analyze the expression of the frog HNF-3β gene. In frog embryos, HNF-3β expressed in the dorsal lip, pharyngeal endoderm and floor plate. In contrast to mammalian HNF-3β, the onset of frog HNF-3β expression in neural tissue occurs after neural tube closure. Thus, the combined expression patterns of Pintallavis and HNF-3β in frogs is equivalent to that of HNF-3β in rats and mice. Within neural tissue, the onset of expression of these regulatory genes define successive stages in the differentiation of floor plate cells. The results reported here show that closely related members of the HNF-3/fork head gene family are expressed by axial midline cell groups involved in neural induction and pattering and suggest the involvement of these genes in the development of the vertebrate neuraxis.