The Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser‐287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Φ‐X‐β‐X‐X‐(S/T)*, where * indicates the phosphorylated residue, Φ is a hydrophobic residue (M>I>L>V), β is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress‐response protein kinases which phosphorylate target proteins with related specificities.