CTCF is a highly conserved, ubiquitously expressed zinc-finger protein with diverse regulatory functions, binding to tens of thousands of the CTCF target sites (CTS) in the genome. CTCF interacts with a number of proteins including the largest subunit of RNA Polymerase H (LS Pol II), previously studied in our laboratory. Two sites within the CTCF Cterminal domain (Site 1 and Site 2) were shown to be involved in the CTCF-LS Pol II binding. The main aim of this study is to further characterize CTCF-LS Pol II interaction and its role in the regulation of CTCF function in transcription. To achieve this aim, seven mutant variants of CTCF, designed to be deficient for binding with the LS Pol II, were generated and characterized. They were then employed in functional assays to assess the implications of the CTCF-LS Pol II interaction in the regulation of CTCF known functions. The interaction of the wild type CTCF (wt CTCF) and the mutant variants with LS Pol II in vivo was investigated using an imaging technique, AP-FRET. All CTCF mutant variants were verified by sequencing and revealed similar stability, localization and molecular weights as the wt CTCF. The CTCF-LS Pol II interactions appeared to be involved in the regulation of the promoteriess luciferase gene fused to the CTSs (alone or in the context of the p14ARF promoter); mutants in the CTCF Site 2 or both sites interfered most with the luciferase activity. However, the effects of this interaction were not confirmed in the context of the endogenous genes, where the role of CTCF-LS Pol II association may be obscured by other influences. In AP-FRET experiments, two mutant variants of Site 1 (lA and IB) and one mutant variant of Site 2 (2A2B), were found to most likely be deficient for interaction with the LS Pol II.