In Drosophila, pulses of the steroid hormone ecdysone function as temporal signals that trigger the major postembryonic developmental transitions. The best characterized of these pulses activates a series of puffs in the polytene chromosomes as it triggers metamorphosis. A small set of early puffs is induced as a primary response to the hormone. These puffs encode regulatory proteins that both repress their own expression and activate a large set of late secondary response genes. We have used Northern blot analysis of RNA isolated from staged animals and cultured organs to study the transcription of three primary response regulatory genes, E75, BR-C and EcR. Remarkably, their patterns of transcription in late larvae can be defined in terms of two responses to different ecdysone concentrations. The class I transcripts (E74B and EcR) are induced in mid-third instar larvae in response to the low, but increasing, titer of ecdysone. As the hormone concentration peaks in late third instar larvae, these transcripts are repressed and the class II RNAs (E74A, E75A and E75B) are induced. The BR-C RNAs appear to have both class I and class II characteristics. These data demonstrate that the relatively simple profile of a hormone pulse contains critical temporal information that is transduced into waves of primary response regulatory gene activity.