Exposure of the immortalized human breast epithelial cell line MCF10A to the Jun N-terminal kinase (JNK) inhibitor anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) suppressed, in a concentration-dependent manner (IC50 is approximately 2 microM), the induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with SP600125 also suppressed the accumulation of TCDD-induced nuclear aryl hydrocarbon receptor (AhR)-DNA complexes, as assessed by electrophoretic mobility shift assays. Concentrations of SP600125 < or = 50 microM did not transform the AhR into a DNA-binding species when added to rat liver cytosol. However, addition of SP600125 to cytosol just before TCDD addition completely suppressed AhR transformation and DNA binding (IC50 approximately 7 microM). Sucrose gradient analyses using rat liver and murine hepatoma 1c1c7 extracts demonstrated that SP600125 competed with TCDD for binding to the AhR. These results suggest that SP600125 is an AhR ligand and functions as an AhR antagonist at concentrations used to pharmacologically inhibit JNK.