We recently identified a novel cDNA encoding a retinoblastoma protein (pRb)-associated protein It was named RBP95, which was composed of 838 amino acid residues with a calculated molecular size of 94,789 Da. Northern blot analysis showed a single mRNA of about 4.5 kb ubiquitously expressed in human tissues. RH mapping results showed that RBP95 is mapped to chromosome region 16p11.2-11.1. Sequence analysis indicated that RBP95 contains a conserved pRb-binding motif LXCXE. Interaction between pRb and RBP95 was confirmed in vivo and in vitro. This interaction requires the LXCXE motif of RBP95 and the entire pocket region of pRb, Each point-mutant of the conserved amino acid residues in pRb-binding motif of RBP95 would destroy its interaction with pRb. RBP95 also contains a basic region leucine zipper and could homodimerize through its leucine zipper region. RBP95 was located in the nucleus with a special pattern when expressed as a GFP fusion in HeLa cells. All these findings suggested that RBP95, a new member of pRb-associated protein, may function as a regulation factor in the process of RNA polymerase II-mediated transcription and/or transcriptional processing.