We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluorescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early G1-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.