Cell growth arrest and apoptosis are two best-known biological functions of tumor-suppressor p53. However, genetic evidence indicates that not only is p21 the major mediator of G(1) arrest, but also it can prevent apoptosis with an unknown mechanism. Here, we report the discovery of a p53 target gene dubbed killin, which lies in close proximity to pten on human chromosome 10 and encodes a 20-kDa nuclear protein. We show that Killin is not only necessary but also sufficient for p53-induced apoptosis. Genetic and biochemical analysis demonstrates that Killin is a high-affinity DNA-binding protein, which potently inhibits eukaryotic DNA synthesis in vitro and appears to trigger S phase arrest before apoptosis in vivo. The DNA-binding domain essential for DNA synthesis inhibition was mapped to within 42 amino acid residues near the N terminus of Killin. These results support Killin as a missing link between p53 activation and S phase checkpoint control designed to eliminate replicating precancerous cells, should they escape G(1) blockade mediated by p21.