The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is involved in a wide range of critical cellular events in response to endogenous signals or xenobiotics. 2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD) is one of the AhR ligands with the maximum affinity for AhR. TCDD is the most toxic among the dioxin xenobiotics and induces a wide range of biological responses, including immunotoxicity and cancer. The complex ligand:AhR:ARNT functions as a transcription factor, binding to the dioxin responsive element (DRE) sequences in the regulatory regions of target genes. Macrophages are key regulators of the innate immune response and being present in all body organs and tissues, they are one of the cell types that are the first to encounter xenobiotics; thus, the insight into the TCDD effect on macrophages is of paramount importance. Putative DREs are predicted using the SITECON software tool in the regulatory regions of the genes encoding transcription factors REL, RELA, and IRF1, expressed in macrophages. The nuclear extract and total RNA are isolated from the U937 macrophage-like cells exposed to 10 nM TCDD (or 0.1% DMSO as the control) for 1, 3, and 6 h. The binding of the TCDD:AhR:ARNT transcription complex from the nuclear extract with double-stranded oligonucleotides containing the putative DREs was studied by the EMSA. Quantitative real-time PCR demonstrates that the expression of these genes in the U937 macrophages increases in a statistically significant manner 1 h (the characteristic time of the maximal dioxin:AhR:ARNT translocation to the nucleus) after exposure to 10 nM TCDD. These results confirm the functional activity of the DREs residing in the IRF1, REL, and RELA regulatory regions via the AhR signaling pathway.