The endothelial cell lines EA.hy926 and HMEC were plated in 3.0 mm (2610 6 pores/cm 2 ) polyester transwell inserts (Corning) and cultured as described above. After reaching 100% confluence, as assessed by lack of penetration of the fluorescent dye FITCdextran 40,000 (and loss of penetration of the L. biflexa serovar Patoc), the monolayer was washed with PBS and cell medium without antibiotics was added to the inserts and wells. Inserts without cells were used as controls for these experiments. Bacteria were added to an MOI of 50 to allow reliable enumeration of bacteria crossing the cell layers or membranes without cells at early time points, and 10 mL from the insert and from the well were taken after 1 h, 3 h, 6 h, 24 h, 27 h, 48 h and 72 h. In addition to the non-pathogenic strain Patoc and the pathogenic Canicola, Leptospira interrogans serovar Copenhageni was also used to analyze the migration of leptospires through the cell monolayer. Motile leptospires were counted by dark-field microscopy using a Petroff-Hausser chamber. Data are shown for the time points through which the bacteria were motile; after 72 hr there was a progressive decrease in L. biflexa motility.