The human Organic Anion Transporter 1 (hOAT1) is a proximal tubular organic anion exchanger of broad specificity that is involved in the elimination of anionic drugs via the kidneys. We have shown previously that the positive residue, arginine (R) at position 466, in OAT1, when exchanged for a negatively charged aspartate (D), not only results in low p-aminohippurate (PAH) transport with decreased affinity but also abolishes the ability of the transporter to interact with glutarate. In this study we performed a more subtle mutation by creating the mutant R466K with a conserved charge replacement. Mutants were created by site directed mutagenesis and functionally characterized by expressing them in Xenopus laevis oocytes using radiolabeled substrates. We observed a significantly lower PAH uptake with R466K hOAT1. However, transport was still qualitatively similar to wild type hOAT1 with respect to affinity; cis-inhibition by other substrates; and trans-stimulation by the dicarboxylate, glutarate. Interestingly, as opposed to the wildtype, which is stimulated by chloride, transport by R466K turned out to be independent of chloride. Thus, the residue arginine, at position 466 appears to be involved in chloride-dependent regulation of hOAT1. Since lysine cannot replace arginine in this aspect the size of the positively charged residue at position 466 is crucial for interaction with chloride.