A 110K-dalton phosphoprotein previously was isolated from the nucleoli of mouse ascites sarcoma cells. The localization of this phosphoprotein in the nucleoli was confirmed by an indirect immunofluorescence assay with rabbit antisera to the phosphoprotein. This phosphoprotein formed a complex of 280K daltons with a nonphosphoprotein of 32K daltons in a molar ratio of 1 to 1. The protein complex dissociated in the presence of 6 M guanidine hydrochloride or 1% sodium dodecylsulphate. The nucleolus-specific phosphoprotein complex bound preferentially to nucleolar DNAs other than the ribosomal RNA gene in vitro and located in nucleosomes prepared from the nucleoli. The major phosphoamino acid in the phosphoprotein was phosphoserine, and slight though significant amounts of phosphotyrosine and phosphothreonine also were detected. These phosphorylated amino acids were concentrated in a specific polypeptide fragment of about 30K daltons obtained by partial digestion with V8 protease. The phosphoprotein was phosphorylated in vitro by the protein kinase activity presented in the complex itself.