The effect of human plasma fibronectin on the adherence of Strepotococcus pyogenes to hexadecane droplets was investigated. Fibronectin blocked the adherence of streptococci to hexadecane in a dose-dependent manner. The inhibitory effect resulted from the binding of fibronectin to the streptococcal cells; radiolabeled fibronectin failed to bind to the hexadecane but bound readily to untreated streptococci. Chemical treatments of streptococci that decreased streptococcal binding of fibronectin also decreased their binding to hexadecane. Pretreatment of fibronectin with lipoteichoic acid blocked the binding of fibronectin to streptococci and abolished its ability to inhibit streptococcal adherence to hexadecane in a dose-related manner. In contrast, wheat germ agglutinin, which binds to N-acetylglucosamine on the surface of S. pyogenes cells, failed to alter hexadecane adherence. The data suggest that fibronectin binds to lipoteichoic acid on the surface of the streptococci, thereby preventing lipoteichoic acid from interacting with the hexadecane phase.