Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.