A screen for Saccharomyces cerevisiae temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. The mutation changed Ser276 to Cys. This amino acid is in the highly conserved NAD(+) binding pocket of the Sir2 family of proteins. Haploid strains of either mating type carrying the mutation were severely defective at mating at 37 degrees but normal at 25 degrees . Measurements of RNA from the HMR locus demonstrated that silencing was lost rapidly upon shifting the mutant from the low to the high temperature, but it took >8 hours to reestablish silencing after a shift back to 25 degrees . Silencing at the rDNA locus was also temperature sensitive. On the other hand, telomeric silencing was totally defective at both temperatures. Enzymatic activity of the recombinant wild-type and mutant Sir2 protein was compared by three different assays. The mutant exhibited less deacetylase activity than the wild-type protein at both 37 degrees and 25 degrees . Interestingly, the mutant had much more NAD(+)-nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD(+) binding pocket of the protein are able to carry out cleavage of NAD(+) to nicotinamide but are defective at the subsequent deacetylation step of the reaction.