The stem cell-specific factor Oct-4 is expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down regulated upon RA-induced differentiation. Irrespective of the direction of differentiation, Oct-4 repression in P19 EC cells requires treatment with high doses of either all-trans or 9-cis RA. Unlike in P19 cells, no RA-induced down regulation of Oct-4 expression is observed in the P19-derived RA-resistant RAC65 cells. However, in these cells Oct-4 promoter repression can be rescued in a RA-dependent manner by cotransfection of RAR alpha 2 or RAR beta 2 but not RARr gamma 1, matching previously reported transactivation properties of these receptor types. In the vicinity of the transcription initiation site of the Oct-4 gene, three Hormone Response Element HRE half sites are present which are arranged as direct repeats with different spacing. In vitro translated RAR and RXR proteins bind to this HRE as heterodimers with low affinity, in such a way that all three HRE half sites contribute to complex formation. Although P19 EC cells contain weak binding activity interacting with the Oct-4 promoter HRE, strong binding activity is observed in nuclear extracts from RA-treated P19 cells. This binding activity was shown to correspond to COUP-TFs but not nuclear RA receptors. Moreover, the presence of these binding factors in nuclear extracts corresponds to silencing of Oct-4 expression. These results implicate RA and the action of its nuclear receptors in silencing Oct-4 expression upon differentiation of EC cells. The observed silencing is most likely not exerted by direct binding of RARs to the Oct-4 proximal promoter HRE. Our results support models in which different nuclear receptor complexes sequentially occupy different sites in the Oct-4 promoter HRE to silence Oct-4 expression during RA-induced differentiation.