Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin.