This study addresses the contributions of speci®c retinoid receptors during all-trans-retinoic acid (RA)-mediated dierentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic eects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARg over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal dierentiation while RARa and RARb do not and RARg overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-R1) having deregulated RARg expression. Since RARg transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 dierentiation. Through gain of function experiments, this report highlights RXRb as playing an important role along with RARg in signaling dierentiation of NT2/D1 cells. Stable over-expression of RXRb, but not RXRa or RXRg, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARg and RXRb resulted in marked growth suppression and for the ®rst time, expression of typical neuronal markers of NT2/D1 dierentiation. To clarify the role of RXRb and RARg in this dierentiation program, a modi®ed transient ®broblast growth factor-4 (FGF4) promoter-enhancer reporter assay that re¯ects eective RA-mediated dierentiation of NT2/D1 cells was employed. Transfection of RARg or RXRb in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARg and RXRb co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 dierentiation. Taken together, these ®ndings reveal speci®c retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the eective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative eects on the dierentiation program of these tumor cells.