Binding of the N-terminus of fibronectin to assembly sites on the cell surface is an essential step in fibronectin fibrillogenesis. Fibronectin matrix assembly sites have customarily been quantified using an iodinated 70 kDa N-terminal fibronectin fragment. The 125I-70 K fragment is a less than ideal reagent because its preparation requires large amounts of plasma fibronectin and it has a fairly short shelf life. An additional limitation is that the cells responsible for binding the 125I-70 K cannot be quantified or identified directly but must be assessed in parallel cultures. To overcome these disadvantages, we developed an ELISA-based assay using a recombinant HA-tagged 70 K fragment. This assay allows for the simultaneous quantification and localization of matrix assembly sites on the surface of adherent cells.