We 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 alpha (RXRalpha). As2O3 did not inhibit transactivation of the estrogen receptor or the glucocorticoid receptor, which do not heterodimerize with RXRalpha. We further show that As2O3 inhibits expression of several target genes of RXRalpha partners. Phosphorylation of RXRalpha has been reported to inhibit nuclear receptor signaling, and we show by in vivo labeling and phosphoamino acid detection that As2O3 phosphorylated RXRalpha 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 RXRalpha phosphorylation and blocked the effects of As2O3 on RXRalpha-mediated transcription. A mutational analysis indicated that phosphorylation of a specific serine residue, S32, was primarily responsible for inhibition of RXRalpha-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.