In teleosts and amphibians, the proneuronal domains, which give rise to primary-motor, primary-inter and Rohon-Beard (RB) neurons, are established at the beginning of neurogenesis as three longitudinal stripes along the anteroposterior axis in the dorsal ectoderm. The proneuronal domains are prefigured by the expression of basic helix-loop-helix (bHLH) proneural genes, and separated by domains (inter-proneuronal domains) that do not express the proneural genes. Little is known about how the formation of these domains is spatially regulated. We have found that the zebrafish hairy- and enhancer of split-related (Her) genes her3 and her9 are expressed in the inter-proneuronal domains, and are required for their formation. her3 and her9 expression was not regulated by Notch signaling, but rather controlled by positional cues, in which Bmp signaling is involved. Inhibition of Her3 or Her9 by antisense morpholino oligonucleotides led to ectopic expression of the proneural genes in part of the inter-proneuronal domains. Combined inhibition of Her3 and Her9 induced ubiquitous expression of proneural and neuronal genes in the neural plate, and abolished the formation of the inter-proneuronal domains. Furthermore, inhibition of Her3/Her9 and Notch signaling led to ubiquitous and homogeneous expression of proneural and neuronal genes in the neural plate, revealing that Her3/Her9 and Notch signaling have distinct roles in neurogenesis. These data indicate that her3 and her9 function as prepattern genes that link the positional dorsoventral polarity information in the posterior neuroectoderm to the spatial regulation of neurogenesis.