Plasma fibronectin has been implicated as an important determinant of neutrophil adhesion to plastic surfaces. Using a monoclonal antifibronectin antibody, we examined the role of fibronectin (Fn) in chemotactic factor-mediated neutrophil attachment to various substrates. The chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP) significantly enhanced neutrophil adherence to multiple substrates including gelatin, gelatin coated with Fc fragments of human IgG or Fn, plastic alone, plastic coated with Fc fragments, or purified plasma Fn. An IgM monoclonal antibody to plasma Fn significantly inhibited FMLP-stimulated neutrophil attachment to gelatin, gelatin-Fc, gelatin-Fn, plastic, plastic-Fc, and plastic-Fn substrates when compared with the parent line myeloma supernatant or an irrelevant IgM monoclonal antibody. No reduction in FMLP-stimulated adherence to the gelatin-plasma or plastic-plasma substrates occurred in the presence of antibody. Anti-Fn antibody reduced FMLP-stimulated adhesion only when present during the entire assay; incubation of cells or substrates alone with antibody, followed by removal of excess antibody before addition of stimulus incubation, failed to alter adherence. These data suggest that neutrophil-derived Fn may play a role in chemotactic factor-induced neutrophil adherence to both collagenous and noncollagenous substrates. Further support for the hypothesis was suggested by the demonstration of release of immunoreactive Fn into incubation media from FMLP-stimulated neutrophils.