Carboxyfluoroquinolones, such as ciprofloxacin, are used for the treatment of numerous infectious diseases. Renal secretion is a major determinant of their systemic and urinary concentration, but the specific transporters involved are virtually unknown. In vivo studies implicate the organic anion transporter (OAT) family as a pivotal component of carboxyfluoroquinolone renal secretion. Therefore, this study identified the specific renal basolateral OAT(s) involved, thereby highlighting potential sources of carboxyfluoroquinolone-drug interactions and variable efficacy. Two heterologous expression systems, Xenopus laevis oocytes and cell monolayers, were used to determine the roles of murine and human renal basolateral mOat1/hOAT1 and mOat3/hOAT3. Ciprofloxacin was transported by mOat3 in both systems (K m value, 70 Ϯ 6 M) and demonstrated no interaction with mOat1 or hOAT1. Furthermore, ciprofloxacin, norfloxacin, ofloxacin, and gatifloxacin exhibited concentration-dependent inhibition of transport on mOat3 in cells with inhibition constants of 198 Ϯ 39, 558 Ϯ 75, 745 Ϯ 165, and 941 Ϯ 232 M, respectively. Ciprofloxacin and gatifloxacin also inhibited hOAT3. Thereafter, in vivo elimination of ciprofloxacin was assessed in wild-type and Oat3 null mice [Oat3(Ϫ/Ϫ)]. Oat3(Ϫ/Ϫ) mice exhibited significantly elevated plasma levels of ciprofloxacin at clinically relevant concentrations (P Ͻ 0.05, male mice; P Ͻ 0.01, female mice). Oat3(Ϫ/Ϫ) mice also demonstrated a reduced volume of distribution (27%, P Ͻ 0.01, male mice; 14%, P Ͻ 0.01, female mice) and increased area under the concentration-time curve (25%, P Ͻ 0.05, male mice; 33%, P Ͻ 0.01, female mice). Female Oat3(Ϫ/Ϫ) mice had a 35% (P Ͻ 0.01) reduction in total clearance of ciprofloxacin relative to wild type. In addition, putative ciprofloxacin metabolites were significantly elevated in Oat3(Ϫ/Ϫ) mice. The present findings indicate that polymorphisms of and drug interactions on hOAT3 may influence carboxyfluoroquinolone efficacy, especially in urinary tract infections.Ciprofloxacin is a broad-spectrum antimicrobial that is used in the treatment of numerous infectious diseases, including those afflicting the skin (Lipsky et al., 1999) (Gogos et al., 1991). It is also a preferred agent for the prevention and treatment of anthrax (Meyerhoff et al., 2004). Its mechanism of action is through the effective inhibition of DNA gyrase, thus preventing DNA replication in susceptible bacteria (Gellert et al., 1977;Sugino et al., 1977). Ciprofloxacin is a carboxylic acidcontaining fluoroquinolone (carboxyfluoroquinolone) that undergoes renal and hepatic elimination, with ϳ50% (oral) or ϳ80% (intravenous) appearing in the urine as parent compound and metabolites 24 h after administration in humans (Höffken et al., 1985). Approximately 20 to 40% of circulating Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.042853.ABBREVIATIONS: LLC-PK 1 , porcine kidney cells; AUC, area under the concentration-t...