The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.