The retinoblastoma protein (pRb) functions as a critical master regulator in cell cycle regulation, which is an important cell-regulatory process, through its interaction with various cellular proteins. Using the C-terminus of human pRb and the yeast two-hybrid system, a novel protein named RBP21 that contains 187 amino acid residues with a calculated molecular weight of 21 kDa was identified as a pRb-binding protein. Sequence analysis indicates that RBP21 shares homology with other retinoblastoma-binding proteins in the pRb-binding motif LxCxE at the C-terminal region. In vitro specific interaction between pRb and RBP21 was confirmed using in vitro translation products. When overexpressed in COS-7 cells, RBP21 could co-immunoprecipitate with pRb. This interaction requires the LxCxE motif of RBP21 and the entire pocket region of pRb. Each point mutation of the conserved amino acid residues in pRb-binding motif of RBP21 abolished its specific interaction with pRb. RH mapping result showed that this novel gene was mapped to chromosome region 15q21.1-21.3. Northern blot analysis suggested that RBP21 was widely expressed in various human tissues and cancer cell lines. When expressed in HeLa cells as a green fluorescent protein fusion, RBP21 was distributed throughout the cell.