ABSTRACT The zebrafish locus one-eyed pinhead (oep) is essential for the formation of anterior axial mesoderm, endoderm and ventral neuroectoderm. At the beginning of gastrulation anterior axial mesoderm cells form the prechordal plate and express goosecoid (gsc) in wild-type embryos. In oep mutants the prechordal plate does not form and gsc expression is not maintained. Exposure to lithium, a dorsalizing agent, leads to the ectopic induction and maintenance of gsc expression in wild-type embryos. Lithium treatment of oep mutants still leads to ectopic gsc induction but not maintenance, suggesting that oep acts downstream of inducers of dorsal mesoderm. In genetic mosaics, wild-type cells are capable of forming anterior axial mesoderm in oep embryos, suggesting that oep is required in prospective anterior axial mesoderm cells before gastrulation. The oep gene is also essential for endoderm formation and the early development of ventral neuroectoderm, including the floor plate. The loss of endoderm is already manifest during gastrulation by the absence of axial-expressing cells in the hypoblast of oep mutants. These findings suggest that oep is also required in lateral and ventral regions of the gastrula margin. The sonic hedgehog (shh).gene is expressed in the notochord of oep animals. Therefore, the impaired floor plate development in oep mutants is not caused by the absence of the floor plate inducer shh. This suggests that oep is required down-stream or in parallel to shh signaling. The ventral region of the forebrain is also absent in oep mutants, leading to severe cyclopia. In contrast, anterior-posterior brain patterning appears largely unaffected, suggesting that underlying prechordal plate is not required for anterior-posterior pattern formation but might be involved in dorsoventral brain patterning. To test if oep has a wider, partially redundant role, we constructed double mutants with two other zebrafish loci essential for patterning during gastrulation. Double mutants with floating head, the zebrafish Xnot homologue, display enhanced floor plate and adaxial muscle phenotypes. Double mutants with no tail (ntl), the zebrafish homologue of the mouse Brachyury locus, display severe defects in midline and mesoderm formation including absence of most of the somitic mesoderm. These results reveal a redundant function of oep and ntl in mesoderm formation. Our data suggest that both oep and ntl act in the blastoderm margin to specify mesendodermal cell fates.