We have previously demonstrated that fibronectin mediates streptococcal adhesion to host cells and that streptococci interact primarily with the N-terminal domain of fibronectin. FBP54 is a 54-kDa protein from group A streptococci that binds fibronectin. In this report, we show that the N-terminal domain of fibronectin reacts with FBP54 and preferentially blocks streptococcal adhesion to buccal epithelial cells. FBP54 blocked adhesion to human buccal epithelial cells by 80% in a dose-related fashion. In contrast, FBP54 had little effect on adhesion of group A streptococci to HEp-2 tissue culture cells. The fibronectin-binding domain of FBP54 has been localized to the first 89 N-terminal residues of the protein. Experiments using affinity-purified antibodies to this region indicated that the N terminus of FBP54 is exposed on the surface of streptococci in a manner that can interact with immobilized receptors. Analysis of sera from patients with post-streptococcal glomerulonephritis and acute rheumatic fever indicated that FBP54 is expressed in vivo and is immunogenic in the human host. These data indicate that FBP54 is a streptococcal adhesin that is expressed in the human host and that preferentially mediates adhesion to certain types of human cells.