Casein kinase II (CKII) of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', that are encoded by the CKA1 and -2 genes, respectively. We have constructed conditional alleles of the CKA1 gene. In contrast to cka1 cka2(ts) strains, which exhibit a defect in both G1 and G2/M cell cycle progression, cka1(ts) cka2 strains continue to divide for three cell cycles after a shift to restrictive temperature and then arrest as a mixture of budded and unbudded cells with a spherical morphology. Arrested cells exhibit continued growth, a nonpolarized actin cytoskeleton, delocalized chitin deposition, and a significant fraction of multinucleate cell bodies, confirming the presence of a cell polarity defect in cka1(ts) strains. The presence of budded as well as unbudded cells in the arrested population suggests that CKII is required for maintenance rather than establishment of cell polarity, although a role in both processes is also possible. The terminal phenotype of cka1(ts) strains bears a strong resemblance to that of orb5 strains of Schizosaccharomyces pombe, which carry a temperature-sensitive CKII catalytic subunit mutation, but the underlying mechanism appears to be different in the two cases. These results establish a requirement for CKII in cell polarity in S. cerevisiae and provide the first evidence for functional specialization of CKA1 and -2.