Histone deacetylation by Saccharomyces cerevisiae Rpd3 represses genes regulated by the Ash1 and Ume6 DNA-binding proteins. Rpd3 exists in a small 0.6 MDa (Rpd3S) and large 1.2 MDa (Rpd3L) corepressor complex. In this report, we identify by mass spectrometry and MudPIT the subunits of the Rpd3L complex. These included Rpd3, Sds3, Pho23, Dep1, Rxt2, Sin3, Ash1, Ume1, Sap30, Cti6, Rxt3 and Ume6. Dep1 and Sds3, unique components of Rpd3L, were required for Rpd3L integrity and HDAC activity. Similar to RPD3, deletion of DEP1 enhanced telomeric silencing and derepressed INO1. Two sequence-specific repressors, Ash1 and Ume6, were stably associated with Rpd3L. While both of these proteins localized to the INO1 and HO promoters, the repression of these genes were dependent only on Ume6 and Ash1, respectively. Thus, the Rpd3L complex is directly recruited to specific promoters through multiple integral DNA-binding proteins.