Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.