The interactions of levofloxacin, a pyridonecarboxylic acid antibacterial drug, with the organic cation transport systems expressed in a pig kidney epithelial cell line, LLC-PK1, were examined. The transcellular transport of tetraethylammonium was remarkably inhibited by levofloxacin, accompanied by a marked increase in the cellular accumulation of tetraethylammonium in the LLC-PK1, monolayers grown on collagen-coated membrane filters. The results obtained by efflux and uptake of tetraethylammonium revealed that levofloxacin drastically inhibited the apical transport activity rather than the basolateral uptake of tetraethylammonium. Under conditions in which the apical efflux of tetraethylammonium was blocked by pretreatment with p-chloromercuribenzene sulfonate, levofloxacin showed a moderate inhibitory effect against the basolateral uptake of tetraethylammonium. Transepithelial flux of levofloxacin from the basolateral side to the apical side was much greater than the flux in the opposite direction. The flux of levofloxacin was influenced by the apical side pH, resulting in a decreased cellular accumulation by lowering pH. The basal-to-apical transport and cellular accumulation of levofloxacin were not inhibited by either tetraethylammonium or cimetidine. These results suggested that levofloxacin interacts with the apical H+/organic cation antiport system to a greater extent than with the basolateral system. However, transcellular transport of levofloxacin would be mediated by the transport systems which are distinct from the systems for tetraethylammonium in LLC-PK1 cells.