The budding yeast genome contains transcriptionally repressed domains at mating-type and telomeric loci, and within rDNA repeats. Gene silencing at telomeres requires the Silent information regulators Sir2p, Sir3p, and Sir4p, whereas only the Sir2p histone deacetylase is required for rDNA repression. To understand these silencing mechanisms biochemically, we examined the subunit structure of Sir2p-containing complexes. Sir2p alone forms a stable homotrimer, whereas the SIR complex is a heterotrimer containing one copy of each Sir protein. A point mutation in the Sir2p core domain (sir2 P394L ) compromises selectively rDNA repression. This mutation impairs homotrimerization but allows SIR heterotrimer formation. Surprisingly, when sir2 P394L is coexpressed with wild-type Sir2p, rDNA repression increases and homotrimers form. Furthermore, coexpression of sir2 P394L and enzymatically inactive sir2 H364Y allows crosscomplementation of rDNA repression defects. The correlation of genetic and biochemical complementation argues that Sir2p trimerization is physiologically relevant for rDNA silencing.