Transcriptional gene silencing controls the expression of repetitive and homologous sequences by cytosine methylation, histone H3 methylation and chromatin modification. TGS suppressor mutants like ddm1, met1 and cmt3 impaired DNA methylation, chromatin assembly and the activity of endogenous genes, transgenes and transposons. But not all modifiers of transcriptional gene silencing affect the same targets. Increasingly there are novel TGS components like MOM1, BRU1, FAS1, FAS2 and RPA2 that are dispensable for DNA methylation. This work aimed at the identification and characterization of new TGS-deficient mutants in Arabidopsis thaliana. For this purpose a new experimental system for analysis transcriptional gene silencing was used for mutant isolation. This system contains a transgenic construct of four in series arranged LUCIFERASE copies for inducing LUCIFERASE silencing and allows the visualization of TGS inhibition after EMS mutagenesis by LUCIFERASE activity. With the help of this system 9 TGS suppressor mutants with significant reactivation of LUCIFERASE silencing could be characterized. Seven mutants showed defects in DDM1, MET1 or CMT3. Here, for the first time, a dominant negative ddm1 mutant could be described. Two further mutants showed significant reactivation of LUCIFERASE silencing in a DNA-methylation-independent manner. For the first time in Arabidopsis thaliana the large subunit of the RIBONUCLEOTIDE REDUCTASE (RNR1) could be connected with transcriptional gene silencing. Interestingly, like BRU1, FAS1, FAS2 and RPA2, RNR1 connects the TGS machinery with DNA replication, repair and chromatin assembly. For the second TGS-deficient mutant independent for DNA methylation a successful analyse could not be concluded due to the genomic localization of the mutation in the pericentric region of chromosome 5.