Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms. In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication. Recent findings suggest that centrin also functions elsewhere in the cell. To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype. Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins. Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function. Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p. A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function. Mutations in the C-terminal region were also defective for interaction with Kic1p. Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.