AbstractThe β‐thymosins are a family of small proteins originally isolated from the thymus. Recently, two of the major mammalian isoforms, thymosin β4 (Tβ4) and thymosin β10 (Tβ10), are identified as significant actin monomer sequestering proteins which may be involved in regulating actin filament assembly. To study the cellular function of β‐thymosins, we have used isoform‐specific antibodies to determine their concentration and intracellular distribution, and examined the effects of inducing overexpression of Tβ4 and Tβ10 on actin filament structures. Immunofluorescence labeling of peritoneal macrophages showed that both β‐thymosins are uniformly distributed within the cytoplasm. cDNA‐mediated overexpression of β‐thymosins in CV1 fibroblasts induced extensive loss of phalloidinstained actin stress fibers. Stress fibers in the cell center were more susceptible than those at the periphery. There was a decrease in the number of focal adhesions, as evidenced by a decrease in discrete vinculin staining and an increase in diffuse vinculin fluorescence. The majority of the transfected cells had normal shape in spite of extensive loss of actin filaments. Occasionally, cells overexpressing β‐thymosin were observed to divide. In these cells, β‐thymosin was excluded from the midbody which contains an actin filament‐rich contractile ring. Our results indicate that Tβ4 and Tβ10 are functionally very similar and both are effective regulators of a large subset of actin filaments in livin