Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.