The substrate specificity of protein kinase-B alpha (PKBalpha, also known as RAC kinase or Akt) was investigated using synthetic peptide substrates related to the sequence surrounding the phosphorylation site on glycogen synthase kinase-3 (GSK3). The minimum sequence motif required for efficient phosphorylation was Arg-Xaa-Arg-Yaa-Zaa-Ser/Thr-Hyd, where Xaa is any amino acid, Yaa and Zaa are small residues other than glycine and Hyd is a bulky hydrophobic residue (Phe, Leu). The most effective substrate, Arg-Pro-Arg-Thr-Ser-Ser-Phe, was phosphorylated with a Km of 5 microM and Vmax of 260 U/mg. PKBalpha phosphorylated histone H2B (Km 5 microM, Vmax 68 U/mg) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. The peptide Arg-Pro-Arg-Ala-Ala-Thr-Phe may be a relatively specific substrate for PKBalpha because, unlike other substrates, it is not phosphorylated by p70 S6 kinase or MAP kinase activated protein (MAPKAP) kinase-1.