The chemical synthesis of thymosin beta 4 using a solid-phase procedure has been accomplished. The synthetic product was found to be homogeneous on paper electrophoresis at pH 6.5, high-performance liquid chromatography on a reversed-phase column, and isoelectric focusing using polyacrylamide gels. The synthetic material was also shown to be identical with the natural thymosin beta 4 by tryptic peptide mapping, amino acid compositional analyses, and polyacrylamide gel isoelectric focusing. Biologically, synthetic thymosin beta 4 was found to be as active as the natural compound in a terminal deoxynucleotidyltransferase induction assay and in a macrophage migration inhibition assay. The proposed structure of the peptide hormone was thus confirmed by a chemical synthesis.