Cells of Staphylococcus aureus strain ISP 546, previously shown to express high binding of vitronectin (Vn), were shown also to bind radioactive iodine-labeled human lactoferrin (Lf). The binding was specific, and heat treatment of S. aureus cells abolished Lf binding. When cells were preincubated with heparin (2.5 mu g/ml), binding of Vn and Lf was inhibited by more than 90%. Two other sulfated carbohydrate polymers, fucoidan and dextran sulfate (mel weights 5000 and 8000) inhibited binding of both iodine-labeled Vn and Lf by about 75% each. No synergistic inhibitory effect between heparin and fucoidan on Lf binding by S. aureus was detected. Mannose, mannoseamine and poly-alpha-sialic acid (colominic acid) inhibited Vn binding by about 40% but had no effect on Lf binding. A cell surface extract of ISP 546 inhibited Vn binding but did not affect Lf binding. Lf binding to S. aureus was inhibited by preincubation of the cells with Vn, and Vn binding to S. aureus was inhibited by preincubation with Lf. Our data indicate that Vn and Lf bind to different molecules on the S. aureus cell surface, involving heparin and other carbohydrate interactions.