Steroid hormones act as important developmental switches, and their nuclear 
receptors regulate many genes. However, few hormone-dependent enhancers have 
been characterized, and important aspects of their sequence architecture, 
cell-type-specific activating and repressing functions, or the regulatory roles 
of their chromatin structure have remained unclear. We used STARR-seq, a 
recently developed enhancer-screening assay, and ecdysone signaling in two 
different Drosophila cell types to derive genome-wide hormone-dependent 
enhancer-activity maps. We demonstrate that enhancer activation depends on 
cis-regulatory motif combinations that differ between cell types and can predict 
cell-type-specific ecdysone targeting. Activated enhancers are often not 
accessible prior to induction. Enhancer repression following hormone treatment 
seems independent of receptor motifs and receptor binding to the enhancer, as we 
show using ChIP-seq, but appears to rely on motifs for other factors, including 
Eip74. Our strategy is applicable to study signal-dependent enhancers for 
different pathways and across organisms.