Abstract Proper development of the midline cells within the Drosophila central nervous system is controlled by the single-minded ( sim) gene. The sim protein defines a new subclass of bHLH transcription factors and as such, is predicted to function as a transcriptional regulator of other genes important for midline development. Here we identify two potential targets of sim regulation: the sim gene itself, and the 47F gene, which is expressed specifically in the sim-expressing midline cells. Using a transgene containing sim regulatory sequences fused to β-galactosidase, we show that the sim gene uses two distinct promoters with overlapping temporal specificities, and that expression from the late promoter is autoregulated by sim itself. By expressing sim protein in salivary gland nuclei using the heat-shock inducible Hsp70 gene promoter, we show that sim protein associates with the sim late promoter on polytene chromosomes. In addition, sim protein binds to a small number of additional chromosomal sites, among which are the sites of the 47F gene, and two other genes, Tl and cdi, whose expression also depend on sim function.