Protein p120 is a proliferation-related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase. Most human malignant tumors contain much higher levels of protein p120 than normal resting cells. To identify p120-associated protein(s), a yeast two-hybrid screen was carried out using protein p120 as the bait. Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58). MSP58 mRNA is 1.9 kb and encodes an approximately 58-kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins. The mouse MSP58 homolog has 97% amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome. The MSP58 N-terminal region contains serine-rich clusters and its C-terminal region has a coiled-coil domain. In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two-hybrid assay. Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled-coil domain within the N-terminal region of p120 and the C-terminal region of MSP58 protein. Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus. Anti-MSP58 Ig labeled nucleolar 'caps' when HeLa cells were treated with actinomycin D. When the MSP58 protein was overexpressed in COS-7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus.