Thymosin beta(4) is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of T beta(4) in actin assembly in the high range of concentrations (up to 300 mu M) at which T beta(4) is found ire highly motile blood cells. T beta(4) behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 mu M). As the concentration of T beta(4) increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The T beta(4)-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and T beta(4)-actin complex, the critical concentration is the sum of free G-actin and T beta(4)-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by T beta(4)-G-actin complex. The incorporation of T beta(4)-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, T beta(4) is not a simple passive actin-sequestering agent, and at high concentrations the ability of T beta(4)-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins