Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.