We recently identified neurogenin (ngn), a neuroD-related bHLH gene, whose Xenopus homolog functions as a neuronal determination factor and upstream activator of XneuroD (Ma et al. Cell 87: 43-52, 1996). Here we identify two additional ngn's, ngn2 and ngn3, which together define a novel subfamily of atonal-related mouse genes. Comparative analysis of ngn expression indicates that these three genes define distinct progenitor populations in the developing CNS and PNS, exhibiting nonoverlapping expression in some areas and partial overlap in others. The expression of the ngn's spatially overlaps and often temporally precedes that of neuroD, suggesting that (as in Xenopus) the ngn's and neuroD function in a cascade. Thus, as in myogenesis, different bHLH determination factors may activate a common bHLH differentiation factor in different sublineages. The ngn's therefore represent both a family of putative mammalian neuronal determination genes and useful markers of the origins of neuronal diversity.