Ofloxacin, a pyridonecarboxylic acid derivative, was examined for its effects on the transport of tetraethylammonium (cation), cephalexin and cephradine (zwitterions), p-aminohippurate (anion) and D-glucose in brush-border membrane vesicles isolated from rat renal cortex. The initial uptake of tetraethylammonium in the presence or absence of an outward H+ gradient was inhibited by ofloxacin in a dose-dependent manner, although the equilibrium value of tetraethylammonium uptake was not affected. This inhibition occurred in a competitive manner (Ki = 0.11 mM). Ofloxacin also inhibited the initial uptake of cephalexin and cephradine, which can be transported via the H+/organic cation antiport system in renal brush-border membranes. In contrast, ofloxacin had no effect on p-aminohippurate. These data suggest that ofloxacin interacts with the organic cation transport system in renal brush-border membranes, and this system may play an important role in the tubular secretion of ofloxacin.