More than 50 ecdysteroid-regulated Drosophila genes have been described in the literature. These genes were identified using several different ecdysteroid-responsive systems and characterized under a variety of experimental conditions. The diversity of these approaches has made it difficult to compare results and identify common responses to the hormone. As a first step toward characterizing the temporal regulation of these genes by ecdysteroids, we have examined their transcriptional activity throughout third instar larval and prepupal development using a single collection of staged animals. We see four coordinate changes in ecdysteroid-regulated gene activity during third instar larval development, at 78-88 hr, approximately 100 hr, 106-108 hr, and approximately 114 hr after egg laying. A dramatic transition in gene expression occurs at puparium formation, after which the prepupal ecdysteroid pulse induces successive waves of transcription. Our results suggest that several increases in the ecdysteroid titer during third instar larval and prepupal development program a precise temporal progression of gene activity that could direct the appropriate behavioral and developmental changes at the onset of metamorphosis.