Silencing in Saccharomyces cerevisiae is found at the mating-type loci HMR and HML, in subtelomeric regions, and at the rDNA locus. Repressed chromatin is built up by the recruitment of the Sir proteins via their interaction with DNA-binding proteins that bind to silencers. Here, we have performed a genetic screen for novel sequence elements within the yeast genome that display silencing activity. We isolated as a novel silencer element the origin of replication from the endogenous 2mu plasmid (2mu ARS). 2mu ARS-mediated silencing was dependent upon the Sir proteins, the origin recognition complex (ORC), and Hst3, a Sir2 histone deacetylase homolog, suggesting that it constituted a novel class of silencing in yeast. Moreover, 2mu ARS carried a binding site for Mig1, a transcriptional repressor of glucose-regulated genes. Both the Mig1-binding site and the MIG1 gene were necessary for full silencing activity of 2mu ARS. Furthermore, Hst3 was physically present at 2mu ARS in a silencing context as well as at the endogenous 2mu plasmid. Also, Hst3 regulated the repression of the flipase gene, although this was likely an indirect effect of HST3 on FLP1 expression.