Members of a subclass ofhairy/Enhancer of split[E(spl)] homologs, calledhesrgenes, are structurally related to another subclass ofhairy/E(spl)homologs,Hesgenes, which play an important role in neural development. To characterize the roles ofhesrgenes in neural development, we used the retina as a model system.In situhybridization analysis indicated that allhesrgenes are expressed in the developing retina, but onlyhesr2expression is associated spatially with gliogenesis. Each member was then misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates, which well mimicin vivoretinal development. Interestingly,hesr2but nothesr1orhesr3promoted gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopted the glial fate by misexpression ofhesr2. The gliogenic activity ofhesr2was more profound when it was misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genesMash1andMath3, which increases Müller glia at the expense of bipolar cells, upregulatedhesr2expression. These results indicate that, among structurally relatedhesrgenes, onlyhesr2promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation betweenhesr2andMash1–Math3may determine the ratios of neurons and glia.