Rapid high-performance liquid chromatographic (HPLC) procedures have been used to isolate and characterize thymosin beta 4 from different species. Crude extracts termed thymosin fraction 5A were prepared from porcine and ovine thymus glands as well as murine spleen. Each fraction 5A preparation was then fractionated by HPLC on a muBondapak C18 reversed-phase column. Porcine and ovine thymus fraction 5A, and murine spleen 5A, each yields a predominant peak at a retention time similar to that of bovine thymosin beta 4. Amino acid analysis as well as HPLC tryptic peptide mapping of these peaks indicate that they have homologous sequences to bovine thymosin beta 4. Chromatographic analysis of fresh murine thymus and spleen tissues also revealed protein peaks at the position of bovine beta 4, suggesting that thymosin beta 4 is the native protein present in the animal tissues.