The retinoblastoma susceptibility gene product (pRB) has been known to function as a negative regulator of cell growth. Recent observations suggest that its biological activity might be modulated by an interaction with nuclear structures. By using in vitro binding assays, we have found that pRB can associate with lamin A, which has been known to be one of the major nuclear matrix proteins. A series of GST-lamin A deletion mutants was constructed to define the amino acid sequence required for binding to pRB. A GST-lamin A (247-355) contained an activity to associate with pRB, while the other constructs, such as GST-lamin A (37-244) or GST-lamin A (356-571), could not bind to pRB. Within the pRB-binding domain of lamin A, there exists the short amino acid sequence which is also present in the pRB-binding region of the transcription factor E2F-1. The similar experiments using a set of GST-RB deletion mutants revealed that a region containing the E1A-binding pocket B and the carboxy-terminal portion of pRB was responsible for binding to lamin A.