The histone acetyltransferase Sas2 is part of the SAS-I complex and acetylates lysine 16 of histone H4 (H4 K16Ac) in the genome of Saccharomyces cerevisiae. Sas2-mediated H4 K16Ac is strongest over the coding region of genes with low expression. However, it is unclear how Sas2-mediated acetylation is incorporated into chromatin. Our previous work has shown physical interactions of SAS with the histone chaperones CAF-I and Asf1, suggesting a link between SAS-I mediated acetylation and chromatin assembly. Here, we find that Sas2-dependent H4 K16Ac in bulk histones requires passage of the cells through the S-phase of the cell cycle, and the rate of increase in H4 K16Ac depends on both CAF-I and Asf1, whereas steady-state levels and genome-wide distribution of H4 K16Ac shows only mild changes in their absence. Furthermore, H4 K16Ac is deposited in chromatin at genes upon repression, and this deposition requires the histone chaperone Spt6, but not CAF-I, Asf1, HIR or Rtt106. Altogether, our data indicate that Spt6 controls H4 K16Ac levels by incorporating K16-unacetylated H4 in strongly transcribed genes. Upon repression, Spt6 association is decreased, resulting in less deposition of K16-unacetylated and therefore in a concomitant increase of H4 K16Ac that is recycled during transcription.