Nucleolin (105 kDa) is the most abundant nucleolar phosphoprotein in exponentially growing cells. We have demonstrated that nucleolin has an intrinsic protease activity for autodegradation. This self-cleaving activity is markedly decreased when cells enter the proliferative stage, resulting in significant stabilization of the nucleolin molecule. The fusion protein derived from a partial nucleolin cDNA clone, which encodes the C-terminal two-thirds of nucleolin, maintained the self-cleaving activity similar to that of the nucleolin purified from nonproliferating cells. The enzymatic domain is therefore located within this region. An exogenous peptide, highly negatively charged, exerted an inhibitory effect, indicating that the enzymatic activity can be regulated. The cleavage kinetics of nucleolin isolated from proliferating cells was apparently concentration independent, suggesting that the autoproteolytic reaction of nucleolin in cells at dividing stage is an intramolecular event. There were many cleavage fragments, which were clustered at four major sites, with apparent molecular weights of about 100, 70, 60, and 50 kDa. These results imply that the nucleolin molecule has repeated motifs that serve as the cleavage sites. Since the detailed electrophoresis patterns, including location and intensity, of the in vitro self-cleaved products of nucleolin derived from nonproliferating and proliferating cells were not quite the same, preferential usage of the particular cutting sites according to cell stage was suggested. It is possible that post-translational modification of nucleolin in proliferating cells may cause these variations.