Fibronectin ("cold-insoluble globulin") has been suggested to play a role in cell-to-cell and cell-to-substratum adhesions. The 70-kilodalton terminal part of human fibronectin has recently been shown to bind to Staphylococcus aureus. In the present study, a fibronectin-binding protein was purified from sonicated S. aureus strain E2371 by affinity chromatography on fibronectin-Sepharose. The fibronectin-binding protein was isolated from an extract of sonicated S. aureus containing at least 57 different proteins as determined by crossed immunoelectrophoresis in antibodies to sonicated S. aureus. The fibronectin-binding protein was released from fibronectin-Sepharose by carbamide (8 M). No impurities in the final preparation could be detected when tested in crossed immunoelectrophoresis. By polyacrylamide gel electrophoresis in both reduced and unreduced gels, the protein showed two bands with relative molecular masses of 197,000 and 60,000, respectively. A complex between the purified S. aureus protein and fibronectin could be demonstrated by crossed immunoelectrophoresis both in monospecific antibodies against fibronectin and in S. aureus polyspecific antibody.