To investigate the allosteric effects of ligands in the function of nuclear receptors, we performed exhaustive alanine scanning mutational analysis (ASMA) of the residues lining the ligand-binding pocket (LBP) of the human vitamin D receptor. The effects of ligands were examined in this system (termed two-dimensional (2D) ASMA) using 10 structurally and biologically characteristic ligands that included agonists, partial agonists, and a full antagonist. The results clearly revealed the role and importance of all the amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D ASMA indicated ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network through which information from ligands is transmitted to the interfaces with protein cofactors and which was shown to be linked to part of the network found by statistical coupling analysis.