Abstract: In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA (RDN1). Silencing in the rDNA is unusual in that Pol II promoters within RDN1 are repressed by Sir2, but not Sir3 or Sir4. Ribosomal DNA silencing unidirectionally spreads leftward, but the mechanism of limiting the spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and Ty1 LTR, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.