Previous studies showed that Staphylococcus aureus expresses a collagen-binding MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecules), CNA, that is necessary and sufficient for S. aureus cells to adhere to cartilage and is a virulence factor in experimental septic arthritis. We have now used a monoclonal antibody (mAb) approach to further analyze the structure and function of CNA. 22 mAbs raised against the minimal ligand binding domain, CNA-(151-318), were shown to bind to the MSCRAMM with similar affinity. All mAbs appear to recognize conformation-dependent epitopes that were mapped throughout the CNA-(151-318) domain using a chimeric strategy where segments of CNA are grafted on ACE, a structurally related MSCRAMM from Enterococcus faecalis. These mAbs were able to inhibit (125)I-collagen binding to CNA-(151-318) as well as to intact S. aureus cells. They also interfered with the attachment of bacteria to collagen substrates. Furthermore, some of the mAbs could effectively displace (125)I-collagen bound to the bacteria. These displacing mAbs were also able to detach bacteria that had adhered to a collagen substrate in a preincubation, raising the possibility that some of the mAbs may be used as therapeutic agents.