Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the wound-healing process. An imbalance in the expression of these molecules is thought to contribute to the failure of chronic ulcers to heal. We investigated whether a mitogenic bovine whey extract enriched with growth factors modulated the expression and activity of MMP-2 and -9, and the tissue inhibitor of MMP-2 (TIMP-2) in chronic leg ulcers. Wound fluids and biopsies were collected from chronic leg ulcer patients whose ulcers were treated topically for 4 weeks with placebo or mitogenic bovine whey extract at concentrations of 2.5, 10, and 20 mg/mL. The levels of MMP-2 and -9 in wound fluid samples was assessed by gelatin zymography and showed a decrease in active MMP-2 in the 2.5 and 10.0 mg/mL mitogenic bovine whey extract-treated ulcers compared with placebo (p<0.05). Immunohistochemical analysis of ulcer biopsies for MMP-2, -9, and TIMP-2 expression showed a reduction in the number of MMP-2-positive dermal fibroblasts in the mitogenic bovine whey extract-treated ulcers compared with pretreatment biopsies (p<0.05) that persisted over the course of the study. In contrast, a transient increase in the number of MMP-9- and TIMP-2-positive cells was observed in mitogenic bovine whey extract treated ulcer biopsies compared with pretreatment levels (p<0.05). These results show that topical application of mitogenic bovine whey extract was able to modulate the expression of MMP-2, -9, and TIMP-2 in chronic leg ulcers and that its constituent growth factors may have the potential to redress the proteolytic imbalance observed in nonhealing chronic ulcers.