Previously, it has been shown that differentiation of embryonal carcinoma (EC) cells turns on the expression of functional transforming growth factor type-beta receptors. Here, we show that the type II receptor (TbetaR-II) gene is activated at the transcriptional level when EC cells differentiate. We show that the differentiated cells, but not the parental EC cells, express transcripts for TbetaR-II. In addition, the expression of TbetaR-II promoter/reporter gene constructs are elevated dramatically when EC cells differentiate and we identify at least two positive and two negative regulatory regions in the 5' flanking region of the TbetaR-II gene. Moreover, we identify a cAMP response element/activating transcription factor site that acts as a positive cis-regulatory element in the TbetaR-II promoter, and we demonstrate that the transcription factor ATF-1 binds to this site and strongly stimulates the expression of the TbetaR-II promoter/reporter gene constructs when ATF-1 is overexpressed in EC-derived differentiated cells. Equally important, we identify a negative regulatory element in a 53-base pair region that had previously been shown to inhibit strongly the expression of TbetaR-II promoter/reporter gene constructs. Specifically, we demonstrate that this region, which contains an inverted CCAAT box motif, binds the transcription factor complex NF-Y (also referred to as CBF) in vitro. Furthermore, expression of a dominant-negative NF-YA mutant protein, which prevents DNA binding by NF-Y, enhances TbetaR-II promoter expression. Together, these studies suggest that the transcription factors ATF-1 and NF-Y play important roles in the regulation of the TbetaR-II gene.