Fibroblasts cultured within free-floating collagen gels can bind to and reorganize the surrounding collagen fibrils into a more dense and compact arrangement. Collagen gel contraction provides an in vitro model for studying fibroblast-collagen interactions important in wound healing, fibrosis, scar contraction, and connective tissue morphogenesis. We have assessed the role of fibronectin and its interaction with the α5β1 “high affinity” fibronectin-specific integrin receptor in collagen gel contraction. A variety of agents, which specifically inhibit fibronectin-α5β1 interactions, were tested for their abilities to inhibit fibroblast-mediated collagen gel contraction. These included anti-α5β1 monoclonal antibodies, the synthetic peptide GRGDSP, the cell adhesive fragment of fibronectin, and an antibody against the cell adhesive region of fibronectin. None of these agents inhibited collagen gel contraction. Therefore, it is concluded that fibronectin-α5β1 interactions are not necessary for collagen gel contraction. However, collagen gel contraction is dependent on a member or members of the β1 subfamily of integrin matrix receptors. A polyclonal antiserum and a monoclonal antibody, both directed against the β1 subunit of integrin matrix receptors, inhibited the spreading of fibroblasts in the collagen gel and inhibited collagen gel contraction. This study demonstrates that fibroblast-mediated collagen gel contraction is independent of fibronectin-α5β1 interactions but dependent on an interaction of β1 integrin matrix receptors with collagen fibers.© Willey-Liss, Inc.