When human umbilical vein endothelial cells (HUVEC) differentiate into capillary-like tubes, there is a five-fold upregulation of the mRNA for thymosin beta4 (Tbeta4) (Grant et al. J Cell Sci 1995; 108: 3685-94 [1]) and this endogenous expression plays an important role in endothelial cell attachment to and spreading on matrix components. We now show that exogenous addition of thymosin beta4 (in the ng-microg range) to HUVEC in culture can induce several biological responses. These responses include increased tube formation in vitro. Additionally, exogenous thymosin beta4 enhances vascular sprouting in the coronary artery ring angiogenesis assay. Measurements of these vascular sprouts show a doubling of the vessel area (via increased branching) with as little as 100 ng of synthetic thymosin beta4. These processes appear to involve the binding of thymosin beta4 to an unknown cell surface receptor and internalization of the protein. This cell surface-binding appears not to be mediated through the thymosin beta4-actin binding domain LKTET. An increase in thymosin beta4 cytoplasmic staining in HUVEC exposed 10 microg of the peptide appears to occur without increased mRNA translation. In summary Tbeta4 induces an increase in cell-matrix attachment, proliferation, tube formation, internalization of the peptide and rearrangement of the actin cytoskeleton. The data now defines both an autocrine and paracrine role for thymosin beta4 in vessel formation.