Thymosin [}4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin [}9 and thymosin [}9 et are minor variants of thymosin [}4. The possible function of these peptides has been investigated by comparing the actin binding properties of these [}-thymosins. Thymosin [}9 and thymosin [}~,t were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 p,M (as compared to 2 q-0.3 12,M for thymosin [}4); like thymosin [}4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin [}4 and thyrnosin [}~,et with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin 134 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin [}4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of -,~ 1.5 btM -I s -I and 2 s -I respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin [}4 function in vivo is discussed.