Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56(chk1). DNA damage induced by ultraviolet light, gamma radiation, or a DNA-alkylating agent has now been shown to result in phosphorylation of p56(chk1). This phosphorylation decreased the mobility of p56(chk1) on SDS-polyacrylamide gel electrophoresis and was abolished by a mutation in the p56(chk1) catalytic domain, suggesting that it might represent autophosphorylation. Phosphorylation of p56(chk1) did not occur when other checkpoint genes were inactive. Thus, p56(chk1) appears to function downstream of several of the known Schizosaccharomyces pombe checkpoint gene products, including that encoded by rad3(+), a gene with sequence similarity to the ATM gene mutated in patients with ataxia telangiectasia. The phosphorylation of p56(chk1) provides an assayable biochemical response to activation of the DNA damage checkpoint in the G(2) phase of the cell cycle.