We isolated cDNA clones for the zebrafish gbx2 gene, which is implicated in the establishment of the midbrain–hindbrain boundary (MHB) in other vertebrates. Spatially localized expression of gbx2 was observed at the MHB from 90% epiboly through to the hatching stage. Comparisons with the expression of otx2, wnt1, and krox20 showed that gbx2 is expressed in the anterior hindbrain. Ectopic expression of gbx2 by mRNA injection caused cyclopia or truncation of the fore- and midbrain and severely affected isthmic and cerebellar structures, while hindbrain formation was not significantly affected. At the molecular level, gbx2 suppressed the expression of otx2 in the fore/midbrain, six3 in the anterior forebrain, and MHB-specific genes such as eng2 and wnt1. In contrast, gbx2 did not down-regulate the expression of the hindbrain marker genes. Therefore, gbx2 specifically suppressed the formation of the entire fore/midbrain. Meanwhile, misexpression of otx2 suppressed the expression of gbx2 in the embryonic brain. Abrogation of gbx2 expression with an antisense morpholino oligonucleotide disrupted the midbrain/anterior hindbrain region, and these loss-of-function effects were rescued by activating the Gbx2 protein immediately after the end of gastrulation. Taken together, these results suggest that the zebrafish gbx2 gene is essential for the maintenance of MHB and/or the formation of the isthmic structure during somitogenesis, rather than for the MHB establishment during gastrulation. We also suggest that other factors, including gbx1, is required for the establishment of the MHB during gastrulation. Developmental Dynamics, 2003. © 2003 Wiley-Liss, Inc.