W e have previously published that 2 proven treatments for acute promyelocytic leukemia, As₂O₃ and retinoic acid, can be antagonistic in vitro. We now report that As₂O₃ inhibits ligand-induced transcription of the retinoic acid receptor, as well as other nuclear receptors that heterodimerize with the retinoid X receptor α (RXRα). As₂O₃ did not inhibit transactivation of the estrogen receptor or the glucocorticoid receptor, which do not heterodimerize with RXRα. We further show that As₂O₃ inhibits expression of several target genes of RXRα partners. Phosphorylation of RXRα has been reported to inhibit nuclear receptor signaling, and we show by in vivo labeling and phosphoamino acid detection that As₂O₃ phosphorylated RXRα in the N-terminal ABC region exclusively on serine residues. Consistent with our previous data implying a role for JNK in As₂O₃-induced apoptosis, we show that pharmacologic or genetic inhibition of JNK activation decreased As₂O₃-induced RXRα phosphorylation and blocked the effects of As₂O₃ on RXRα-mediated transcription. A mutational analysis indicated that phosphorylation of a specific serine residue, S32, was primarily responsible for inhibition of RXRα-mediated transcription. These data may provide some insight into the rational development of chemotherapeutic combinations involving As₂O₃ as well as into molecular mechanisms of arsenic-induced carcinogenesis resulting from environmental exposure.