A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408-427)] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.