The pluripotent human embryonal carcinoma (EC) cell line NTERA-2 provides a useful tool for investigating cell differentiation in a way that is pertinent to the development of the early human embryo. The major immediate early (MIE) gene of human cytomegalovirus (HCMV), which is not transcribed in undifferentiated NTERA-2 EC cells but is transcribed in their differentiated derivatives, offers a model with which to study the developmental regulation of gene activity during the differentiation of these cells. We have investigated the regulatory activity of the cAMP response elements (CRE) and the activation protein (AP1) site found within several repeated 19-base-pair (bp) elements from the HCMV MIE promoter, and the developmental regulation of nuclear DNA-binding factors that interact with these sites. The 19-bp CRE but not the AP1 site is responsive to cAMP in undifferentiated NTERA-2 EC and its activity is enhanced upon differentiation. Nuclear proteins of the CREB, Fos, and Jun families bind to these sites, but, surprisingly, their levels only show limited regulation during NTERA-2 differentiation. This contrasts with results obtained with murine EC cells. However, additional and apparently novel proteins with molecular weights between 80,000 and 90,000, and binding specificities for both CRE and AP1 sites, were detected in undifferentiated EC cells. The activity of these proteins decreased markedly after differentiation, indicating their involvement in negative regulation of the CRE/AP1-like site in undifferentiated EC cells. This suggests novel members able to interact via leucine zippers with other members of the Jun-Fos-CREB family of DNA binding proteins that are also involved in this regulation.