The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix transcription factor that heterodimerizes with the aryl hydrocarbon receptor to mediate signal transduction pathways inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and other planar aromatic hydrocarbons. Monoclonal antibodies (MAbs) have been raised against a carboxyl-terminal 19-amino acid peptide hapten (MAb 2B10) and against a carboxyl-terminal 378-amino acid polypeptide-staphylococcal Protein A fusion protein (MAb 4G9) of Arnt and their characterization is described. Western blot experiments show that both MAbs specifically cross-react with an approximately 85-kDa band in cytosol prepared from COS-7 cells transfected with the full length human Arnt cDNA pBMSNeo-D24-1 and in Hepa 1c1c7 cytosol but not in Arnt-deficient Hepa 1-C4 mutant cytosol. Velocity sedimentation of Hepa 1c1c7 cytosol on sucrose gradients and Superose 6 gel permeation chromatography were used to estimate the sedimentation coefficient. Stokes radius, and relative molecular mass of Arnt as approximately 3.6-4.1 S, 6.8 nm, and 101-115 kDa, respectively. These results indicate that Arnt probably exists in monomeric form in Hepa 1c1c7 cytosolic extracts. Laser scanning confocal microscopy and indirect immunofluorescence microscopy revealed Arnt to be distributed throughout the non-nucleolar portion of the nucleus of Hepa 1c1c7, VT(2) (Hepa 1-C4T mutant cell line deficient in Arnt function and stably transfected with pBMSNeo D24-1, expressing the full length human Arnt cDNA), and HeLa cells. The establishment of the nuclear localization of Arnt in human and murine cell lines shown here indicates that its nuclear localization may be conserved across species. Immunofluorescence analysis of Arnt in three cell lines using two MAbs (to distinct epitopes) provides evidence that suggests that the aryl hydrocarbon receptor heterodimerizes with Arnt in the nucleus.