In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.