Two procedures were devised to examine initial events in the glucocorticoid-regulated transcription of integrated murine mammary tumor virus (MTV) DNA sequences. First, cells in monolayer culture were exposed for brief periods to dexamethasone and then permeabilized in situ with digitonin to allow entry of 32P-labeled ribonucleoside triphosphates and measurement of transcription rates. The results revealed that the rate of MTV RNA synthesis is stimulated selectively by dexamethasone after an apparent lag of approximately equal to 1 min and is half-maximal by 8-9 min. Second, the time course of transcription elongation was monitored by quantitating in single strand nuclease protection assays the accumulation of RNA sequences from different regions of the 7.8-kilobase MTV transcription unit. These experiments suggested that the rate of MTV RNA elongation in the presence of hormone does not differ substantially from elongation rates calculated for other genes transcribed by RNA polymerase II. Moreover, dexamethasone increased in parallel and to the same extent the levels of RNA from various segments of the transcription unit, implying that transcripts are elongated across the MTV element at a constant rate. We conclude that glucocorticoids stimulate MTV transcription solely via a rapid and selective increase in the efficiency of transcription initiation.