Activity of the estrogen receptor (ER) is regulated through interaction with coactivators and corepressors. These proteins are present in large complexes, suggesting functional interactions among various cofactors. Scaffold attachment factors B1 and B2 (SAFB1/2) and nuclear receptor corepressor (N-CoR) function as ERalpha corepressors--they directly interact with ERalpha, and repress transcription via repression domains. We asked the question whether SAFB1/2 and N-CoR could directly interact with each other, and whether this interaction results in altered repressive activities. Employing coimmunoprecipitation, cofractionation, and colocalization experiments, we have shown that SAFB1/2 interact with the nuclear receptor corepressor N-CoR. This interaction was direct, and was mediated in vitro and in vivo through the C-terminal region of SAFB1 (amino acids 600-915 and the N-terminal region of N-CoR (amino acids 1-373)). Decrease of SAFB1 or N-CoR expression by small interfering RNA resulted in an increase of the estrogen response in reporter assays, confirming prior data that both proteins are attenuating estrogen-mediated induction of genes. Importantly, the effect of SAFB1 on this attenuation was significantly decreased in the presence of N-CoR small interfering RNA. Using chromatin immunoprecipitation assays, we observed that SAFB1/2 and N-CoR were recruited to the pS2 promoter in the absence of estrogen, and this recruitment was enhanced in the presence of Tamoxifen. Detailed kinetic studies showed that the addition of estrogen resulted in the concurrent release of SAFB1/2 and N-CoR from the promoter. Finally, we measured expression of SAFB1/2 and N-CoR in 289 clinical breast cancer specimens, and detected a strong and highly significant correlation between their expression levels. Taken together, our studies demonstrate that SAFB1/2 and N-CoR interact, and that this interaction is, at least in part, necessary for SAFB1's repressive activities. The coexpression of these proteins in breast cancer specimens, and the combined recruitment (and release) of SAFB1/2 and N-CoR furthermore suggests that this interaction has functional relevance.