The acetylation state of histones plays a central role in determining gene expression in chromatin. The reestablishment of the acetylation state of nucleosomes after DNA replication and chromatin assembly requires both deacetylation and acetylation of specific lysine residues on newly incorporated histones. In this study, the MYST family acetyltransferase Sas2 was found to interact with Cac1, the largest subunit of Saccharomyces cerevisiae chromatin assembly factor-I (CAF-I), and with the nucleosome assembly factor Asf1. The deletions of CAC1 (cac1Delta), ASF1 (asf1Delta), and SAS2 (sas2Delta) had similar effects on gene silencing and were partially overlapping. Furthermore, Sas2 was found in a nuclear protein complex that included Sas4 and Sas5, a homolog of TAF(II)30. This complex, termed SAS-I, was also found to contribute to rDNA silencing. Furthermore, the observation that a mutation of H4 lysine 16 to arginine displayed the identical silencing phenotypes as sas2Delta suggested that it was the in vivo target of Sas2 acetylation. In summary, our data present a novel model for the reestablishment of acetylation patterns after DNA replication, by which SAS-I is recruited to freshly replicated DNA by its association with chromatin assembly complexes to acetylate lysine 16 of H4.