Interactions of Leptospira interrogans with cultured endothelial and kidney epithelial cells were assayed by examining (i) cytoadherence of intrinsically radiolabeled leptospires to eucaryotic cell monolayers and (ii) penetration of leptospires through cell monolayers grown on polycarbonate filters in chemotaxis chambers. L. interrogans serovars attached to cultured cells in a dose- and time-dependent manner. Adherence was diminished following pretreatment of organisms with proteases, rabbit immune serum, or heat. When observed by scanning electron microscopy, most leptospires attached by both ends, rather than just one tip like Treponema pallidum. In penetration assays, 9.7% of added L. interrogans migrated through the monolayer-filter barrier, while only 0.3% of L. biflexa penetrated in the same time interval. Transmission electron microscopy revealed that organisms entered the host cell cytoplasm. These in vitro results indicate that leptospires have an invasive capacity that may be related to pathogenicity in vivo and suggest that further investigation of interactions with host cells may enhance knowledge of leptospiral virulence.