Osteomyelitic rat tibiae were examined by scanning electron microscopy for the extracellular glycocalyx of Staphylococcus aureus. S. aureus and fractured tibiae from normal rats were incubated together in vitro and examined similarly. Low magnification of endosteal Haversian portals from tibiae studied in vivo and in vitro disclosed adherent S. aureus exuding glycocalyx that buried the organism in dense, coccoid-studded biofilms. The biofilm became progressively more dense over time in vitro and was exuberant at day 70 in vivo. S. aureus incubated in vitro without tibiae disclosed no glycocalyx. Bone chips studied in vitro disclosed staphylococci more commonly near the endosteal Haversian portals than on the intervening endosteal surfaces (mean +/- SE, 280 +/- 75 vs. 12 +/- 3 per 2,500-micron 2 field; P less than .002 by Student's t test). Organisms within ostia were not counted, although they occluded 10%-40% of the ostium. Staphylococci were adherent to exposed woven material, perhaps collagen.