Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin β4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β4 × G-actin complex.