The retinoblastoma gene product (pRB) has been known to contain a sequence-nonspecific DNA binding activity. It is unknown whether pRB can recognize and bind to specific DNA sequences. We have recently identified a rat genomic DNA fragment, termed REC11, which can interact with rat Cdc37-related protein (RCdc37). In this study, we have found that pRB could interact with the REC11, DNA in a sequence-specific manner. A series of GST-RB deletion mutants was used in the gel shift assays to define the domain of pRB responsible for this interaction. GST-RB (385-611) and GST-RB (612-928) completely lost the binding activity, while GST-RB (555-682) retained an activity to associate with the REC11 DNA, indicating that the continuous spacer region of pRB might be important for this sequence-specific DNA binding activity.