The question of how proneural bHLH transcription factors recognize and regulate their target genes is still relatively poorly understood. We previously showed that Scute (Sc) and Atonal (Ato) target genes have different cognate E box motifs, suggesting that specific DNA interactions contribute to differences in their target gene specificity. Here we show that Sc and Ato proteins (in combination with Daughterless) can activate reporter gene expression via their cognate E boxes in a non-neuronal cell culture system, suggesting that the proteins have strong intrinsic abilities to recognize different E box motifs in the absence of specialized cofactors. Functional comparison of E boxes from several target genes and site-directed mutagenesis of E box motifs suggests that specificity and activity require further sequence elements flanking both sides of the previously identified E box motifs. Moreover, the proneural cofactor, Senseless, can augment the function of Sc and Ato on their cognate E boxes and therefore may contribute to proneural specificity.