The expression of the E2A transcription unit of the adenovirus E1A deletion mutant dl312 in murine embryonal carcinoma (EC) stem cells suggests that they contain an activity that will complement viral E1A. We have prepared from these cells in vitro transcription extracts that use E1A-inducible promoters more efficiently than do extracts from a differentiated cell line. Mixing experiments demonstrate that the EC phenotype is dominant. Gel retardation assays using the E2A promoter detect a binding activity present in F9 and PCC4 EC cells but not in differentiated cells. Our data indicate that EC stem cells contain a transcription factor that is analogous to viral E1A and is likely to be involved in the control of cellular gene expression during differentiation.