A prominent feature of cell differentiation is the initiation and maintenance of an irreversible cell cycle arrest with the complex involvement of the retinoblastoma (RB) family (RB, p130, p107). We have isolated the HBP1 transcriptional repressor as a potential target of the RB family in differentiated cells. By homology, HBP1 is a sequence-specific HMG transcription factor, of which LEF-1 is the best-characterized family member. Several features of HBP1 suggest an intriguing role as a transcriptional and cell cycle regulator in differentiated cells. First, inspection of the HBP1 protein sequence revealed two consensus RB interaction motifs (LXCXE and IXCXE). Second, HBP1 interaction was selective for RB and p130, but not p107. HBP1, RB, and p130 levels are all up-regulated with differentiation; in contrast, p107 levels decline. Third, HBP1 can function as a transcriptional repressor of the promoter for N-MYC, which is a critical cell cycle and developmental gene. Fourth, because the activation of the N-MYC promoter in cycling cells required the E2F transcription factor, we show that E2F-1 and HBP1 represent opposite transcriptional signals that can be integrated within the N-MYC promoter. Fifth, the expression of HBP1 lead to efficient cell cycle arrest. The arrest phenotype was manifested in the presence of optimal proliferation signals, suggesting that HBP1 exerted a dominant regulatory role. Taken together, the results suggest that HBP1 may represent a unique transcriptional repressor with a role in initiation and establishment of cell cycle arrest during differentiation.