Retinoblastoma protein (pRb) functions as a tumor suppressor, and certain proteins are known to bind to pRb in the C-terminal region. Although the N-terminal region of pRb may also mediate interaction with some proteins, no such protein has been identified yet. We demonstrated previously the in vivo protein association between pRb and 73-kDa heat shock cognate protein (hsc73) in certain human tumor cell lines. In this report we analyzed the interaction between these two proteins in vitro. Our data showed that hsc73 interacts with the novel N-terminal region of pRb; that is, pRb binds directly to hsc73 and dissociates from hsc73 in an ATP-dependent manner. By using deletion mutants of cDNA encoding pRb, the hsc73 binding site of pRb was determined to be located in the region (residues 301-372) outside the so-called A pocket (residues 373-579) of this tumor suppressor protein. This finding was compatible with the fact that the adenovirus E1A oncoprotein, which is known to bind to the E2F binding pocket region of pRb, could not compete with hsc73 for the binding. Furthermore, phosphorylation of pRb by cyclin-dependent kinase inhibited the binding of pRb to hsc73. These data suggest that hsc73 may act exclusively as the molecular chaperone for nonphosphorylated pRb. As a result, hsc73 may function as a molecular stabilizer of nonphosphorylated pRb.