Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in 3-dimensional collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 siRNA were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without TGF-ß1 for five days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a non-specific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9 deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9 deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9 deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-ß1 and reduced several TGF-ß1 driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Since TGF-ß1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-ß1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3-dimensional collagen gels mediated by fibroblasts.