Two major DNA-binding activities specific for the major histocompatibility (MHC) class I regulatory element (CRE) were detected in adenovirus (Ad)-transformed cells. One activity, term CRE1, had similar binding properties to a previously described positive-acting transcription factor specific for MHC class I genes termed H2TF1. The other activity, termed CRE2, bound to a region on the CRE separate from CRE1, and was present in Ad12, but not in Ad5-transformed cells. A CRE2-like activity was also present in non-adenovirus-transformed mouse L929 cells, indicating that CRE2 may be a cellular, rather than a viral, factor. The CRE2 activity did not correspond to any previously described transcription factor with a potential binding site in the CRE.