An in vitro transcription system was developed from H411EC3 (H4) hepatoma cells, which mimics the in vivo up-regulation by glucocorticoid hormones on ribosomal RNA (rRNA) synthesis. Ribosomal DNA (rDNA) transcription in extracts derived from H4 cells grown in the presence of 100 nM triamcinolone acetonide was 4- to 5-fold greater than that in extracts derived from cells grown in the absence of glucocorticoid. This effect was not a general stimulation by the steroid, as RNA polymerase II transcription of the metallothionein-1 gene which lacked a glucocorticoid responsive element was unaffected. The increased transcription in hormone-treated extracts was also independent of differential ribonuclease activities or inhibitors as ascertained by the inclusion of ribonuclease inhibitor and mixing experiments, respectively. Chromatography of H4 cell extracts on heparin-sepharose followed by transcription complementation analysis, showed that the hormone-induced stimulatory activity eluted with the fraction (TFIA) which contains RNA polymerase I (Pol I). Immunoblot analysis with specific anti-Pol I antibody showed similar subunit profiles in the absence and presence of the hormone. The presence of a Pol I enhancer element in addition to the rDNA promoter did not further modify the glucocorticoid-induced transcription. These results indicate that the glucocorticoid-mediated effects could be observed in cell extracts which accurately initiate transcription of cloned rat rDNA. Moreover, the alterations of rDNA transcription by the hormone is effected by a factor which elutes with fraction TFIA.