All eukaryotes use feedback controls to order and coordinate cell cycle events. In Schizosaccharomyces pombe, several classes of checkpoint genes serve to ensure that DNA replication is complete and free of error before the onset of mitosis. Wild-type cells normally arrest upon inhibition of DNA synthesis or in response to DNA damage, although the exact mechanisms controlling this arrest are unclear. Genetic evidence in fission yeast suggests that the dependence of mitosis upon completion of DNA replication is linked to the regulation of the p34cdc2 cyclin-dependent kinase. It has been hypothesized that inhibition of DNA synthesis triggers down-regulation of p34cdc2 kinase activity, although this has never been shown biochemically. We analyzed the activity of p34cdc2 in wild-type and checkpoint-defective cells treated with a DNA synthesis inhibitor. Using standard in vitro assays we demonstrate that p34cdc2 kinase activity is maintained in wild-type cells arrested at the replication checkpoint. We also used a novel in vivo assay for p34cdc2 kinase activity, in which we expressed a fragment of the human retinoblastoma tumor suppressor protein in fission yeast. Phosphorylation of this fragment of the human retinoblastoma tumor suppressor protein is dependent on p34cdc2 kinase activity, and this activity is also maintained in cells arrested at the replication checkpoint. These data suggest that the mechanism for cell-cycle arrest in response to incomplete DNA synthesis is not dependent on the attenuation of p34cdc2 activity.