The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RARalpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RARalpha antagonists do not influence RARalpha/RXRalpha beterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RARalpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RARalpha/RXRalpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RARalpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RARalpha conformation seems to allow RARalpha/RXRalpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RARalpha. Complexation of RARbeta, RARgamma, and RXRalpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.