Fibronectin (FN) is a major extracellular matrix protein involved in various biological events. This study demonstrated that the third FN type III repeat (FnIII3) and several fragments containing the repeat promote cell spreading and migration of human dermal fibroblasts (HDFs), whereas the fourth repeat (FnIII4) did not. A variety of cell types also spread on FnIII3 in a cell-type-specific manner, but not on FnIII4. Immunofluorescence assays revealed that FnIII3 induced the organization of focal contacts and stress fibres in HDFs. Cyclic [RGDFV] peptides with a D-Phe residue, which are selective inhibitors of cell adhesion to vitronectin, inhibited HDF spreading on FnIII3 equally with GRGDS, indicating little involvement of alphaV-integrins in FnIII3 spreading. An anti-beta1 integrin mAb inhibited cell spreading on FnIII3 and FN. To our knowledge, this is the first demonstration that a novel domain of FnIII3 functions in cell spreading and migration through an interaction with unresolved beta1 integrin(s) in an RGD-dependent manner.