Using a two-layer system, a bottom layer containing treponema cells suspended in NOS (New Oral Spirochete)-Noble agar medium or NOS-Bacto agar medium and overlaid with cell-free NOS-agarose medium resulted in the spirochete cells migrating into the top layer. However, if the positions of the medium layers were reversed with the cells inoculated into the bottom layer containing NOS-agarose, there was no migration into the upper layer. This suggests migration of the spirochetes away from Bacto and Noble agars. Using a 3-layer system in which cells were inoculated into a middle layer consisting of NOS-agarose medium and sandwiched between cell-free NOS-agarose medium layers, cells remained within the middle layer. If the cells were inoculated into a middle layer consisting of NOS-Bacto agar medium while the upper and lower layers remained unchanged, cells migrated into both upper and lower layers. If cells that had migrated into the upper layer were transferred into a middle layer, they virtually all migrated into the upper layer repeatedly. Cells that had migrated into the lower layer and transferred to the middle layer migrated repeatedly into the lower layer. These results suggest the possible existence of two distinct locomotory phenotypes within this strain of treponeme.