For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. We investigated the contribution of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). We show that the proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, we define an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus, thereby defining distinct E(Ato) and E(Sc) sites. These E-box variants are crucial for function. First, tandem repeats of 20-bp sequences containing E(Ato) and E(Sc) sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo. Second, interchanging E(Ato) and E(Sc) sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity.