Nuclear receptor corepressor (N-CoR) mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. N-CoR contains several repression domains that repress transcription when fused to a heterologous DNA binding domain, but their relative importance in the full-length N-CoR molecule is unknown. Here we addressed this important issue by depleting N-CoR in human cells and replacing it with mutant and wild-type murine N-CoR. Although the N-terminal RD binds transducin beta-like protein 1 (TBL1), TBLR1, and mSin3, deletion of this region did not affect the ability of N-CoR to mediate repression by TR. By contrast, deletion of the deacetylase activating domain (DAD) that binds and activates histone deacetylase 3 dramatically hampered N-CoR's function as a TR corepressor. Introduction of a single amino acid mutation in the DAD similarly disabled the corepressor function of N-CoR. Thus, the DAD domain of N-CoR is singularly essential for repression by TR.