The 68C region of the Drosophila melanogaster salivary gland polytene chromosomes harbors the structural genes for the three salivary gland glue proteins sgs-3, sgs-7, and sgs-8. This region is puffed during the third larval instar, the stage when glue proteins are being produced in the salivary glands. The puff regresses near the end of the third instar as a result of an increased titer of the steroid hormone ecdysterone in the larval hemolymph. The experiments reported here were designed to determine whether the ecdysterone effect on puffing at 68C is correlated with hormone effects on expression of the three puff RNAs. In the first series of experiments, it is shown that there is a more rapid disappearance of 68C RNA transcripts from salivary glands cultured in the presence of ecdysterone than from glands cultured in its absence. The second set of experiments, in which 68C transcripts were pulse-labeled in salivary glands cultured in the presence or absence of hormone, demonstrates that one effect of ecdysterone is to cause a sharp reduction in the rate at which newly synthesized 68C transcripts accumulate. The final experiments follow the time required for ecdysterone to produce this effect, and show that it occurs in salivary glands exposed to the hormone for as little as 15 min. In all of the experiments, the RNA products of the Sgs-3, Sgs-7, and Sgs-8 genes acted coordinately.