Thymosin β4 (Tβ4) binds to G‐actin in vitro and inhibits actin polymerization. We studied the effects of incresing Tβ4 concentration within living PtK2 cells, comparing its effects on the disassembly of stress fibers and membrane‐associated actin with its ability to inhibit cytokinesis and cell spreading after mitosis. We chose PtK2 cells for the study because these cells have many striking actin bundles in both stress fibers and cleavage furrows. They also have prominent concentrations of membrane‐associated actin and remain flattened during mitosis. We have found that PtK2 cells contain an endogenous homologue of Tβ4 at a concentration (approximately 28 μM) sufficient to complex a third or more of the cell's unpolymerized actin. Intracellular Tβ4 concentrations were increased by three different methods: (1) microinjection of an RSV vector containing a cDNA for Tβ4; (2) transfection with the same vector; and (3) microinjection of purified Tβ4 protein. The plasmid coding for Tβ4 was microinjected into PtK2 cells together with fluorescently labeled alpha‐actinin as a reporter molecule. Immediately after microinjection fluorescently labeled alpha‐actinin was detected in a periodic pattern along the stress fibers just as in control cells injected solely with the reporter. However, after 13 h, cells microinjected with reporter and plasmid showed marked disassembly of the fiber bundles. PtK2 cells transfected with this RSV vector for 2–3 days showed disassembly of stress fibers as detected by rhodamine‐phalloidin staining; in these cells the membrane actin was also greatly diminished or absent and the border of the cells was markedly retracted. Microinjection of pure Tβ4 protein into interphase PtK2 cells induced disassembly of the stress fibers within 10 min, while membrane actin appeared only somewhat reduced. If the PtK2 cells were mitotic, Similar microinjection of pure thymosin β4 protein at times from early prophase to metaphase resulted in an unusual pattern of delayed cytokinesis. Furrowing occurred but at a much slower rate than in controls and the amount of actin in the cleavage furrow was greatly reduced. The cells constricted to apparent completion, but after about 30 min the furrow re‐gressed, forming a binucleate cell, much as after treatment with cytochalasin B or D. Postcytokinesis spreading of these Tβ4‐injected cells was often inhibited. These experiments suggest that an insufficient number of actin filaments prolongs the contractile phase of cytokinesis and abolishes the final sealing process. © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc.