In Saccharamyces cerevisiae, the homothalic mating loci HML and HMR are transcriptionally silenced. Silencing requires both cis-acting silencers (E amd I) flanking the silenced regions, and trans-acting factors including the products of the SIR2 through SIR4 genes. There is overwhelming evidence indicating that transcriptionally silenced chromatin is distinct from active chromatin. We studied the architecture of silenced chromatin at HML by analyzing the topology of DNA spanning the locus. This was accomplished by examining the supercoiling of DNA circles excised from the HML locus. We observed that HML-circles were more negatively supercoiled in a wild type strain than in a sir3- strain in which silencing at HML is abolished. HML-circles without the silencers maintained the high negative supercoiling. Deleting the promoters for the two genes within HML had no effect on the supercoiling. These results establish that, (1) DNA in transcriptionally silenced chromatin assumes distinct topology that is not due to the lack of transcription, and (2) silencers are not required for maintaining the topology. We are testing the hypothesis that the higher negative supercoiling at HML is due to the fact that histones H3 and H4 in the nucleosomes at HML are hypoacetylated, which may affect the wrapping of DNA around the nucleosomal core.