Cell-matrix interactions are of significant importance for tissue homeostasis of the skin and, if disturbed, may lead to ageing and hyperplastic scar formation. We have studied fibroblasts stably overexpressing manganese superoxide dismutase (MnSOD) with a defined capacity for the removal of superoxide anions and concomitant accumulation of hydrogen peroxide to evaluate the role of enhanced MnSOD activity on the dynamics of cell-matrix interactions in the three-dimensional collagen lattice contraction assay. MnSOD overexpressing fibroblast populated collagen lattices revealed a significantly enhanced contraction compared to collagen lattices populated with vector control cells. The enhanced collagen lattice contraction was in part due to an increase in active TGF-beta1 and the accumulation of H2O2 in MnSOD overexpressing fibroblasts populated collagen lattices. Inhibition of TGF-beta1 signalling by the ALK4,5,7 kinases' inhibitor SB431542 at least partly inhibited the enhanced collagen lattice contraction of MnSOD overexpressing fibroblasts populated lattices. In addition, supplementation of vector control fibroblast populated collagen lattices with recombinant TGF-beta1 concentration dependently enhanced the collagen lattice contraction. In the presence of the antioxidant Ebselen, a mimic of H2O2 and other hydroperoxides/peroxynitrite-detoxifying glutathione peroxidase, collagen lattice contraction and the activation of TGF-beta1 were significantly reduced in collagen lattices populated with MnSOD overexpressing fibroblasts. Collectively, these data suggest that H2O2 or other hydroperoxides or peroxynitrite or a combination thereof may function as important second messengers in collagen lattice contraction and act at least in part via TGF-beta1 activation.