Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.