The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix transcription factor family. The AhR interacts with a wide variety of xenobiotic agonists, the most potent of which is 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), to produce a wide variety of biologic and toxic responses such as teratogenicity and cancer. TCDD and other dioxins are emitted from the combustion of fossil fuels, metalsmelting and processing, manufacturing of chemicals, photochemical and biological processing, as well as in food. Exposure from these sources of dioxin may lead to adverse health effects. It has been suggested that AhR activity is regulated by post-translational modifications. Phosphorylation of the AhR is a key regulatory mechanism that plays a role in DNA binding and transcriptional activity; however, few studies have identified functionally significant sites of phosphorylation. These studies investigated possible functional phosphorylation at the STS (serine 393, threonine 394, and serine 395) region of the mouse AhR. This sequence is located in a major region of phosphorylation and in the transcriptional inhibitory domain. Phosphorylation prediction algorithms suggest that serine 393 and serine 395 may be phosphorylated by protein kinase A (PKA). This research was conducted to test the overall hypothesis that the phosphorylation of the STS sequence is important for regulating AhR-mediated transcriptional activity. Studies determined that PKA is able to phosphorylate the full-length receptor as well as peptides containing the STS sequence via ³²P incorporation in vitro. Additionally, Fourier Transform Mass Spectrometry (FTMS) analysis of PKA-treated peptides shows that Serine 395 is phosphorylated. Functionally, we show that there is decreased transcriptional activity when serine 395 is mutated to alanine without affecting binding of the AhR-ARNT (aryl hydrocarbon receptor nuclear translocator) complex to dioxin-response elements. These data suggest that phosphorylation of serine 395 regulates the transactivation ability of the mouse AhR. The results from these experiments identify a functionally significant phospho-site and further confirm that the AhR may be regulated by phosphorylation (Funded by NIH Grants ES02515, ES01247, and ES07025).