To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1 ϩ and spp2 ϩ (S. pombe primase 1 and 2). spp1 ϩ encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Pol␣ to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1 ϩ gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1 ϩ exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1 ϩ reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Pol␣-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Pol␣ has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Pol␣. Together, these have led us to propose that suboptimal cellular levels of pol␣-primase complex due to the allele-specific mutations of Spp1 might not allow Pol␣ to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.