Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn2+, but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg2+, DNA ends, and Bh proteins. From p53 peptide mutagenesis analysis, me found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH2-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.