The attachment of Staphylococcus aureus (Cowan I) and two strains of group A and G streptococci on glass cover slips coated with fibronectin, fibronectin fragments, or fibrinogen was studied. The attachment was quantitated by counting the attached bacteria on glass surfaces coated with a similar molarity of the proteins. Fibronectin was a more effective attachment factor than fibrinogen for staphylococci, while group G streptococci attached better on fibrinogen- than on fibronectin-coated cover slips. In this system, group A streptococci bound almost exclusively to substrate-bound fibrinogen. Attachment experiments involving the use of staphylococci pretreated with soluble fibronectin or fibrinogen revealed that bacterium-bound fibronectin and fibrinogen were able to enhance the adherence on cover slips coated with fibronectin. The 30-kilodalton NH2-terminal and the 120- to 140-kilodalton COOH-terminal fragments of fibronectin, both of which contain bacterial binding sites, mediated the staphylococcal attachment, suggesting that both parts of the molecule are involved in the attachment mediated by fibronectin.