Gene expression can be affected by the chromosomal position of the gene. An example of this position effect is silencing of the HML and HMR mating-type loci of Saccharomyces cerevisiae. An in vitro assay revealed that silencing induced a transcription-independent general occlusion of the DNA at HMR from sequence-specific interactions of proteins with DNA. The minimum boundaries of the silenced chromatin structure were determined, as were the contributions of the E and I silencers to the size of the silenced domain. Examination of endonuclease-sensitive sites provided evidence that neither the integrity of the chromosomal duplex nor covalent linkage of the silencers to HMR was important for maintenance of the silenced structure in vitro.