H uman retinoid X receptor α (hRXRα) is a member of the nuclear receptor family of transcriptional regulators. It regulates transcription through its association with several heterodimeric partners, including the vitamin D₃ receptor (VDR). Signaling through the VDR is essential for normal calcium homeostasis and has been shown to inhibit the proliferation of cancer cells derived from a number of tissues. Here we show that phosphorylation of hRXRα in ras-transformed human keratinocytes through the activated Ras–Raf–mitogen-activated protein kinase (Ras-Raf-MAP kinase) pathway results in attenuated transactivation by the VDR and resistance to the growth inhibitory action of 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] and RXR-specific agonist LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl) ethenyl]-benzoic acid). Phosphorylation of hRXRα occurs at serine 260, a consensus MAP kinase site. Inhibition of MAP kinase activity or point mutagenesis of serine 260 of hRXRα reverses the observed resistance to 1,25(OH)₂D₃ and LG1069. Thus, hRXRα is a downstream target of MAP kinase, and its phosphorylation may play an important role in malignant transformation.J. Clin. Invest. 103:1729–1735 (1999).