Posttranslational acetylation of histones is an important element of transcriptional regulation. The yeast Tup1p repressor is one of only a few non-enzyme proteins known to interact directly with the amino-terminal tail domains of histones H3 and H4 that are subject to acetylation. We demonstrated previously that Tup1p interacts poorly with more highly acetylated isoforms of these histones in vitro. Here we show that two separate classes of promoters repressed by Tup1p are associated with underacetylated histones in vivo. This decreased histone acetylation is dependent upon Tup1p and its partner Ssn6p and is localized to sequences near the point of Tup1p-Ssn6p recruitment. Increased acetylation of histones H3 and H4 is observed upon activation of these genes, but this increase is not dependent on transcription per se. Direct recruitment of Tup1p-Ssn6p complexes via fusion of Tup1p to the lexA DNA binding domain is sufficient to confer repression and induce decreased acetylation of H3 and H4 at a target promoter. Taken together, our results suggest that stable decreases in histone acetylation levels are directed and/or maintained by the Tup1p-Ssn6p repressor complex.