Saccharomyces cerevisiae strains bearing temperature-sensitive alleles of the cell division cycle gene CDC31 are specifically defective in duplication of the spindle pole body, the microtubule-organizing center of yeast. To define the function encoded by CDC31 more fully, we have isolated genomic clones of the gene by selection for complementation of a temperature-sensitive allele. The locus from which the clone was derived was marked by integration of a nutritional marker and found by meiotic mapping to cosegregate with CDC31. The polypeptide sequence of the open reading frame in the CDC31 gene was determined and compared with the sequences of other known proteins. This revealed significant homology with the calmodulins and other members of the Ca2+-binding protein family. On the basis of comparison with these related proteins, it is evident that the CDC31 gene product has at least two binding sites for Ca2+ and is also homologous with other regions of the calmodulin sequence. We propose that Ca2+ fluxes within the yeast cell play a key role in spindle pole body duplication and consequently in the organization of the microtubule arrays.