The amino acid sequence of thymosin beta 4, a polypeptide isolated from calf thymus, was determined. Thymosin beta 4 is composed of 43 amino acid residues and has a molecular weight of 4982 and an isoelectric point of 5.1. The NH2 terminus of the peptide is blocked by an acetyl group. This molecule induces expression of terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase, EC 22.214.171.124) in transferase-negative murine thymocytes in vivo and in citro. Thus, it appears that thymosin beta 4 acts on lymphoid stem cells and may control the early stages of the maturation process of thymus-dependent lymphocytes. This peptide is one of several present in thymosin fraction 5 that participates in the regulation, differentiation, and function of thymus-dependent thymocytes.