The histone deacetylase RPD3 can be targeted to certain genes through its interaction with DNA-binding regulatory proteins. RPD3 can then repress gene transcription. In the yeast Saccharomyces cerevisiae, association of RPD3 with the transcriptional repressors SIN3 and UME6 results in repression of reporter genes containing the UME6-binding site. RPD3 can deacetylate all histone H4 acetylation sites in cell extracts. However, it is unknown how H4 proteins located at genes near UME6-binding sites are affected, nor whether the effect of RPD3 is localized to the promoter regions. Here we study the mechanism by which RPD3 represses gene activity by examining the acetylation state of histone proteins at UME6-regulated genes. We used antibodies specific for individual acetylation sites in H4 to immunoprecipitate chromatin fragments. A deletion of RPD3 or SIN3, but not of the related histone-deacetylase gene HDA1, results in increased acetylation of the lysine 5 residue of H4 in the promoters of the UME6-regulated INO1, IME2 and SPO13 genes. As increased acetylation of this residue is not merely a consequence of gene transcription, acetylation of this site may be essential for regulating gene activity.