Tumor suppressor p53 is known to inhibit transactivation by certain nuclear receptors, and overexpressed p53 is known to correlate with poor differentiation in liver cancer. Therefore, we investigated whether wild-type p53 might also affect the function of hepatocyte nuclear factor 4alpha1 (HNF4alpha1), an orphan receptor required for liver differentiation. Our results show that HNF4alpha1-mediated transactivation is repressed by p53 but that the mechanism of repression is not due to inhibition of HNF4alpha1 DNA binding. Rather, transfections with Gal4 fusion constructs indicate that the repression is via the ligand-binding domain of HNF4alpha1. Furthermore, we found that p53 in human embryonic kidney whole-cell extracts preferentially bound the ligand-binding domain of HNF4alpha1 and that the activation function 2 region was required for the binding. Competition for coactivator CREB binding protein could not entirely account for the repression but trichostatin A, an inhibitor of histone deacetylase activity, could reverse p53-mediated repression of HNF4alpha1. In contrast, p53-mediated repression of transcriptional activation of the same promoter by another transcriptional activator, CCAAT/enhancer-binding protein-alpha, could not be reversed by the addition of trichostatin A. These results suggest that p53, like other transcriptional repressors, inhibits transcription by multiple mechanisms, one of which involves interaction with the ligand-binding domain and recruitment of histone deacetylase activity.