Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.