The adenovirus ElA proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level ElA expression in the absence of viral infection would facilitate biochemical studies of ElA-mediated transactivation. Toward this end, we introduced the adenovirus type 2 ElA gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable ElA mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of ElA expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying ElA concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same ElA concentration and that transcription from each promoter increased as the ElA concentration increased. These results indicate that ElA-mediated transactivation is proportional to the concentration of ElA protein. ElA-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the ElA proteins was sufficient to increase the transcriptional activity of nuclear extracts.