Aqueous humor collected from freshly enucleated bovine eyes was passed over a gelatin affinity column, and bound material was eluted with 4M urea. When fractionated by sodium dodecyl sulfate electrophoresis, the eluted material co-migrated with human plasma fibronectin and protein standards of approximately 220,000 daltons. Amino acid analyses demonstrated a composition similar to that of human plasma fibronectin and fibronectin synthesized by cultured cells. The concentration of fibronectin in aqueous humor is approximately 100 times less than in human plasma.