A set of genes activated by the hormone ecdysone has previously been identified in the salivary glands of wandering Drosophila larvae, along with a group of regulatory elements involved in transcriptional induction by ecdysone. New evidence was obtained to implicate the DNA-binding protein Crol in the transcriptional regulation of ecdysone-dependent genes in larval salivary glands. Crol was shown to bind to the promoters and enhancers of ecdysone-depending genes, demonstrating an increased binding in the salivary glands, where the genes are active, compared to brain tissue, where the genes are silent. Crol was found to prime the recruitment of the ecdysone receptor (EcR) at least on some ecdysone-dependent enhancers. Disruption of Crol binding at these sites in crol knockout larvae reduced the EcR binding level and decreased the transcriptional level of genes possessing the respective regulatory elements. Based on the findings, Crol was assumed to function as one of the many proteins that render regulatory regions susceptible to EcR binding, thereby enabling the specific effect of ecdysone on D. melanogaster tissues.