It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.