The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer activated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related xenobiotics. Ligand binding initiates a series of poorly understood molecular events which confers recognition of cis-acting elements located in regulatory regions of particular structural genes, such as CYP1A1. Several studies have suggested that AhR phosphorylation may be instrumental in activating the AhR to a DNA-binding state. In agreement with previous investigations, treatment of the AhR with acid phosphatase resulted in the loss of DNA-binding activity. To further evaluate the functional role of AhR phosphorylation we determined whether TCDD binding altered total AhR phosphorylation, and identified phosphorylated regions by the examination of chemical cleavage patterns. The AhR was isolated by immunoprecipitation from [32P]-orthophosphate-labeled Hepa 1 cells grown in the presence or absence of TCDD. Examination of the amount of 32P associated with the AhR indicated that the total level of AhR phosphorylation was not affected by ligand binding. Chemical cleavage with hydroxylamine and cyanogen bromide also revealed a similar pattern for liganded and unliganded AhR. The shortest regions of overlap determined by the chemical cleavage patterns localized phosphorylation sites to two regions in the C-terminal half of the AhR. One region is centrally located between amino acids 368 and 605 and within or adjacent to a DNA binding repressor domain. The other region is located at the glutamine-rich carboxyl terminus between amino acids 636 and 759. These data coupled with previous observations imply that total AhR phosphorylation is not altered by the ligand-elicited transformation to a DNA-binding form, but that phosphorylation nevertheless plays an important role in the ability of an active AhR-Arnt complex to associate with cis-acting regulatory elements.