In a previous study, we reported that two synthetic peptides derived from the 33-kD carboxyl terminal cell/heparinbinding fragment of fibronectin A chain promoted keratinocyte adhesion but not spreading. Because keratinocytes are capable of spreading on the 33 66-kD fragments, we focused on identifying additional chemically synthesized peptides from the cell/heparin-binding fragments of fibronectin that might promote cell spreading. When plastic substrata were coated with peptide FN-C/H-V (WQPPRARI), which is derived from the carboxyl-terminal heparin-binding domain of all plasma fibronectin isoforms, keratinocytes adhered and displayed a spread morphology. In solution, soluble peptide FN-C/H-V inhibited cell spreading on intact fibronectin and on the 33 66-kD fragments. Furthermore, polyclonal antibodies raised against peptide FN-C/H-V also inhibited keratinocyte spreading on fibronectin and the 33 66-kD fragments. These data support the hypothesis that keratinocyte cell adhesion and cell spreading on fibronectin are mediated by multiple distinct domains and different regulatory processes.