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The aryl hydrocarbon receptor (AhR) is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but activation without an exogenous ligand also occurs when normal cell–cell contact is prevented. Suspension of several C3H10T1/2 fibroblast clonal sub-lines that contain an integrated AhR-responsive reporter produced a time course and level of reporter activation and CYP1B1 induction that paralleled TCDD stimulation in confluent monolayer culture. Suspension activation was, however, more transient. Loss of cell–cell contact at low density also activated these reporters independent of cell cycle changes to levels comparable to TCDD stimulation of confluent cells. Loss of cell–cell contact may, therefore, activate AhR. Suspension and TCDD activations exhibited comparable nuclear translocation of AhR and then AhR/ARNT complex formation. Each AhR activation process was equally attenuated by inhibition of, respectively, HSP90 ATPase, the 26S proteosome, and by depletion of intracellular Ca2+. By contrast, the AhR antagonist α-naphthoflavone (αNF) blocked ligand-stimulated AhR activity, but not activation through loss of cell–cell contact. Suspension-induced reporter activation was selectively enhanced by LiCl, which prevented GSK-3β effects on the simultaneously released β-catenin. The effects of suspension and LiCl on reporters were reversed by Ro-31-8220, which did not affect β-catenin, TCDD-activation processes, or AhR turnover. Neither LiCl nor Ro-31-8220 altered suspension-induced AhR/ARNT complex formation. Loss of cell–cell contact permits nuclear translocation and AhR activation that is largely replicated after TCDD binding, but with activity differences due to contact-sensitive factors functioning after AhR/ARNT complex formation.