Abstract: In Saccharomyces cerevisiae, silencers flanking the HML and HMR loci consist of various combinations of binding sites for Abf1p, Rap1p and the origin recognition complex (ORC) that serve to recruit the Sir silencing complex thereby initiating the establishment of transcriptionally silent chromatin. There have been seemingly conflicting reports concerning whether silencers function in an orientation-dependent or -independent manner, and what determines the directionality of a silencer has not been explored. We demonstrate that chromatin plays a key role in determining the potency and directionality of silencers. We show that nucleosomes are asymmetrically distributed around the HML-I or HMR-E silencer so that a nucleosome is positioned close to the Abf1p side but not the ORC side of the silencer. This coincides with preferential association of Sir proteins and transcriptional silencing on the Abf1p side of the silencer. Elimination of the asymmetry in nucleosome positioning at a silencer leads to comparable silencing on both sides. Asymmetric nucleosome positioning in the immediate vicinity of a silencer is independent of its orientation and genomic context indicating that it is the inherent property of the silencer. Moreover, it is also independent of the Sir complex and thus precedes the formation of silent chromatin. Finally, we demonstrate that asymmetric positioning of nucleosomes and directional silencing by a silencer depend on ORC and Abf1p. We conclude that the HML-I and HMR-E silencers promote asymmetric positioning of nucleosomes leading to unequal potentials of transcriptional silencing on their sides, and hence directional silencing.