Treponema denticola has been shown to bind to immobilized fibronectin (Fn) by its tips. Yet labeling of cells in suspension with an Fn-gold conjugate to localize the Fn adhesins shows that they are distributed in patches along the entire cell length. Subsequent experiments have shown that the number and proportion of tip-oriented cells increase with time, suggesting that Fn contact stimulates T. denticola to rearrange adhesins toward its tips. To test this hypothesis, T. denticola cells were allowed to migrate in a 2% methylcellulose column toward nitrocellulose filters coated with Fn, laminin, bovine serum albumin, or phosphate-buffered saline. Cells close to and distant from the filters were collected, labeled with Fn-gold probes, and examined by transmission electron microscopy. The number of gold particles on each of 20 cells was counted, dividing each cell into thirds along its length: the end third with the most label (end 1), the middle third, and the end with the least label (end 2). The mean number (+/- standard deviation) of gold probes per third was calculated. Fn-gold probes clustered toward one end of T. denticola cells when in contact with Fn-coated nitrocellulose, with > 55% of probes in end 1. In contrast, no clustering toward T. denticola ends occurred with laminin-, bovine serum albumin, or phosphate-buffered saline-coated filters or in the absence of a filter. Blocking access of the T. denticola cells to the Fn-coated nitrocellulose filter by placing an uncoated filter between them prevented clustering of Fn-gold. Removal of T. denticola cells from direct contact with the Fn-coated filter did not promote redistribution of clustered probes. These data suggest that T. denticola is stimulated to cluster Fn adhesins irreversibly toward its tips when it migrates into contact with immobilized Fn. This might be significant for establishing multiple adhesive interactions with host cells and ligands.