The aryl hydrocarbon receptor (AhR) is known to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxic effects. Immunocytochemical studies revealed that AhR in HeLa cells is localized throughout the cell. Upon TCDD treatment most of the cytoplasmic receptor is translocated into the nucleus in a time-dependent manner. A significant amount of AhR was found to be tightly associated with the nuclear fraction of untreated HeLa cells. The level of receptor in the nuclear fraction was approximately 16% of the total cellular receptor pool. Further characterization of AhR heterocomplex from the HeLa nuclear fraction by sucrose density gradient analysis revealed that the AhR was present in the 6 S form, and that the nuclear AhR could be coimmunoprecipitated using anti-Arnt mAb. The ability of the AhR to specifically interact with dioxin-responsive elements (DRE) was demonstrated utilizing wild-type and two mutant DREs in gel shift assays. These results would suggest that, in HeLa cells, the AhR-Arnt heterodimer is associated with the nuclear fraction under normal culture conditions. Therefore, HeLa cells can be used as a model system to study the biochemical and molecular function of the Ah receptor and the process that leads to activation of the AhR in the absence of exogenous ligand.