Monoclonal antibodies against fibronectin were used to locate the gelatin-binding and cell-attachment regions of plasma fibronectin at an ultrastructural level. A total of 23 hybridomas were generated using mice immunized with either intact fibronectin or a 40 000 Mr gelatin-binding fibronectin fragment. One of these antibodies (D9b) strongly inhibited the interaction of radio-labelled fibronectin with gelatin. Another antibody (IB10) inhibited the attachment of Chinese hamster ovary (CHO) cells to a fibronectin substratum by 99%. Both of these antibodies were purified by affinity chromatography on columns of fibronectin-Sepharose and were then incubated with soluble fibronectin to form antigen-antibody complexes. The complexes were separated from free antibody on a column of Sephadex G-200 and were prepared for electron-microscopic examination by spraying on mica discs and rotary shadowing with platinum. As determined by this method, the fibronectin molecules measured 124 +/- 1.7 nm in length. Monoclonal antibody IB10 was visualized as a globular projection 40 +/- 1.4 nm from one end of the fibronectin filament. Monoclonal antibody D9b, on the other hand, was visualized as a globular projection at or near one or both ends of the molecule. These data provide the first morphological localization of the gelatin-binding and cell-attachment regions of fibronectin and indicate that further studies using monoclonal antibodies directed toward other epitopes should shed light not only on function but also on the tertiary and quaternary structure of the fibronectin molecule.