The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP(-/-) mouse embryonic fibroblasts (MEFs) and AHRR(-/-) MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP(-/-) MEFs, AHRR(-/-) MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR(-/-) MEFs or AHRR Delta 8, the active isoform of AHRR, in TiPARP(-/-) MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRR Delta 8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRR Delta 8_Delta 1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRR Delta 8_Delta 1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRR Delta 8_Delta 1-49 nor GFP-AHRR Delta 8_Delta 1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.