The E74 early ecdysone-inducible gene plays a key role in the regulatory hlerarchy activated by ecdysone at the onset of Drosophlla metamorphosis. We show here that E74A protein binds to three adjacent sites in the middle of the E74 gene. The consensus sequence for E74A protein binding, determined by random-sequence ollgonucleotide selection, contains an invariant purine-rich core sequence, C/AGGAA. This sequence is also present in the binding sites of two mammalian proteins that, like E74A, are related to the ets oncoprotein. Antibody staining of larval sallvary gland polytene chromosomes revealed that E74A protein binds to both early and late ecdysone-inducible puffs. This study supports Ashburner's proposal that the early puffs encode site-specific DNA binding proteins that directly interact with the early and late ecdysone-inducible puffs. © 1990.