By using (35)S-labeled calmodulin (CaM), we have isolated a full-length cDNA clone expressing the Schizosaccharomyces pombe homologue of calmodulin kinase I (CaMK-I), a gene we have named cmk1. It has been previously been shown in mammals that CaMK-I is a member of a CaM-dependent protein kinase cascade that ultimately regulates transcription factors such as ATF and cAMP-response element-binding protein. The cmk1 cDNA encodes a 335-amino acid protein with significant homology to mammalian CaMK-I, including a conserved sequence for phosphorylation by CaM kinase kinase. We have expressed the cmk1 cDNA in bacteria and yeast, and we have shown that it is a CaM-dependent protein kinase. A truncation mutant of cmk1 (d320) failed to bind CaM, indicating that the CaM-binding domain is at the extreme C terminus of the protein. The mRNA for cmk1 is expressed in a cell cycle-dependent manner, peaking at or near the G(1)/S boundary. Overexpression of wild-type cmk1 in S. pombe caused no apparent effects on growth and division. However, mutation of a predicted regulatory site (Thr-192) to aspartic acid resulted in hyperactivation of CMK1 activity in the presence of CaM and causes cell cycle arrest in vivo. Arrest is also accompanied by morphological defects. These results suggest the presence of a CaM-dependent protein kinase cascade in yeast and indicate that cmk1 may be important in cell cycle progression, a process known to be dependent on CaM in eukaryotic cells.