Embryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF-beta 2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF-beta 2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF-beta 2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF-beta 2 gene in the murine parietal endoderm-like cell line, PYS-2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF-beta 2 gene in PYS-2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF-beta 2 gene reduces significantly the activity of the TGF-beta 2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells.(ABSTRACT TRUNCATED AT 250 WORDS)