The expression of human papillomavirus (HPV) E6 oncoprotein is causally linked to high-risk HPV-associated human cancers. We have recently isolated hADA3, the human homologue of yeast transcriptional co-activator yADA3, as a novel E6 target. Human ADA3 binds to the high-risk (cancer-associated) but not the low-risk HPV E6 proteins and to immortalization-competent but not to immortalization-defective HPV16 E6 mutants, suggesting a role for the perturbation of hADA3 function in E6-mediated oncogenesis. We demonstrate here that hADA3 directly binds to the retinoic X receptor (RXR)alpha in vitro and in vivo. Using chromatin immunoprecipitation, we show that hADA3 is part of activator complexes bound to the native RXR response elements within the promoter of the cyclin-dependent kinase inhibitor gene p21. We show that hADA3 enhances the RXR(alpha)-mediated sequence-specific transactivation of retinoid target genes, cellular retinoic acid-binding protein II and p21. Significantly, we demonstrate that E6 inhibits the RXR(alpha)-mediated transactivation of target genes, implying that perturbation of RXR-mediated transactivation by E6 could contribute to HPV oncogenesis.