Pim-1 is an oncogene-encoded serine–threonine kinase that is expressed primarily in cells of the hematopoietic system and germ line. The full-length coding regions of both human and Xenopus laevis Pim-1 were expressed as recombinant bacterial fusion proteins that autophosphorylated in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pim-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ribosomal protein S6 (residues 229–249). The optimal substrate peptide for Pim-1 was determined to be Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-X, where X is an amino acid residue with a small side chain. These results were confirmed using X. laevis Pim-1 expressed in COS cells. These findings could permit the identification of physiological substrates of Pim-1 and predict the location of phosphorylation sites within these proteins.