The 110 kDa component of the yeast spindle pole body (SPB) encoded by the SPC110/NUF1 gene is an essential protein which is important for the generation of a functional mitotic spindle in the yeast Saccharomyces cerevisiae. Spc110p exists in a number of forms which differ in mobility upon separation by SDS-polyacrylamide gel electrophoresis. We show that this heterogeneity is due to differential phosphorylation on serine or threonine residues and that Spc110p phosphorylation varies throughout the cell cycle. Specifically, the phosphorylated form Spc110p appears as cells progress from S-phase and is coincident with the presence of preanaphase mitotic spindles. Thus phosphorylation of Spc110p occurs at the stage in the cell cycle where SPBs acquire the competence to organize a mitotic spindle and could therefore represent an important regulatory event.