Murine F9 embryonal carcinoma (EC) stem cells have an Ela-like transcription activity that is undetectable in F9 cells differentiated to parietal endoderm-like cells (F9-PE). The Ela-inducible adenovirus E2A promoter has been used to further define this activity and we show that in vitro the transcription of this promoter in F9 EC and F9-PE cell extracts reflects the regulation in vivo. In EC cell extracts several trans-acting protein factors bind to E2A promoter sequences. A distal domain containing a CRE binds proteins present in F9 EC, F9-PE and Hela cell extracts. Sequences between -71 and -50 define a multiplicity of binding activities, termed DRTF1, all of which are down regulated as EC stem cells differentiate. DRTF2, a low abundance, regulated binding activity requires DNA sequences that overlap those required by DRTF1. The CRE and the DRTF1 binding site compete for transcription in vitro, indicating that in EC cell extracts the respective proteins function as positively acting, binding site dependent transcription factors. Comparison of DRTF1 with the previously defined HeLa cell factor E2F, induced during adenovirus infection, indicates that although both factors recognise the same region of the promoter there are clear differences between them. These data indicate that multiple factors are necessary for efficient transcription of the E2A promoter in F9 EC cell extracts and suggest that DRTF1 is responsible, at least in part, for the developmental regulation of the cellular Ela-like activity.