Cell cycle-dependent transcriptional repression of the E2F1 and B-myb promoters is mediated through E2F-binding sites and adjacent corepressor site (cell cycle gene homology region (CHR)/downstream repression site (DRS)). Here, we show that a factor binding to the B-myb CHR is co-purified with E2F DNA-binding activity, and coimmunoprecipitated with components of E2F/Rb-family repressor complexes, E2F4 and retinoblastoma (Rb) family proteins. In spite of structural and functional similarities, however, the E2F1 and B-myb CHRs exhibited distinct factor-binding specificities. Furthermore, substitution of E2F1 CHR with the B-myb CHR in the E2F1 promoter revealed that the B-myb CHR was unable to repress the E2F1 promoter completely in the G0 phase. These results suggest that transcriptional repression of the E2F1 and B-myb promoters is mediated by physical interaction of E2F/Rb-family repressor complexes with promoter-specific corepressors. (C) 2007 Elsevier Inc. All rights reserved.