Platelet-associated fibronectin antigen has been identified by radioimmunoassay and immunofluorescent techniques. In radioimmunoassay, platelet fibronectin was immunochemically indistinguishable from plasma fibronectin. Platelet and plasma fibronectin were bound and eluted from gelatin-sepharose under similar conditions. The level of platelet fibronectin in detergent extracts of washed platelets from 12 healthy adults was 2.85 +/- 1.24 microgram/10(9) platelets. Immunofluorescence with F(ab')2 fragments of immunochemically purified antifibronectin showed that all platelets stained with a discrete punctate pattern. The identification of platelet fibronectin antigen raises the possibility that this protein may participate in platelet-platelet or platelet-surface interactions.