We isolated two zebrafish sequences containing a homeobox related to orthodenticle (otd), a gene expressed in the developing head of Drosophila. One of these is clearly homologous to Otx1, a homeobox gene previously reported to be expressed in the developing rostral brain of the mouse. We termed this zebrafish gene otx1. The second gene is not as closely related to Otx1 and is equally divergent from Otx2, a second homeobox gene expressed in the developing rostral brain of the mouse. We termed it otx3, even if a corresponding murine Otx3 gene has not been reported yet. Both genes are expressed in early-gastrula zebrafish embryos in the involuting presumptive anterior mesendoderm. With the extension of the body axis, the expression domain of both genes extends to neuroectodermal regions fated to become fore- and mid-brain. From this stage the expression domains of the two genes differ slightly from each other but both cover the rostral brain with a sharp posterior boundary coinciding with that between midbrain and hind-brain. This late expression closely corresponds to that of the murine Otx1 gene, whereas the earliest expression of both zebrafish otx genes is different from that of Otx1 and reminiscent of that of Otx2 in the mouse. In this light, the zebrafish otx1 and otx3 genes appear to share some expression features of both murine Otx1 and Otx2. It will be of considerable interest to study the specific role of the various genes of the otx family in the development of the zebrafish brain regions. The peculiar spatio-temporal pattern of these genes during early zebrafish gastrulation suggests a role of this gene family in interactions between anterior mesendoderm and neuroectoderm.