Because citrulline plasma concentration is elevated in kidney failure, citrulline could be a biomarker of renal insufficiency, though the mechanism regulating its disposition in kidney has not been clarified. In rat kidney slices, citrulline uptake was apparently Na⁺-dependent, saturable with K_m 556 µM, and significantly inhibited by anionic (PAH) and cationic (TEA) compounds, but not by probenecid at 1 mM. Preincubation of kidney slices with glutarate increased citrulline uptake, while such an increase was not observed after preincubation of the slices in Na⁺-free buffer. This result suggested that a sodium-dependent dicarboxylate cotransporter is involved in citrulline uptake by rat kidney slices. In studies using transporter-overexpressing cells, human organic anion transporter 1 (OAT1) and rat Oat1 exhibited citrulline transport activity with K_m values of 238 µM and 373 µM, respectively, while other OATs and organic cation transporters (OCTs) did not transport citrulline. Based on the relative activity factor method, the contribution of rat Oat1 to the overall uptake of citrulline in rat kidney slices was approximately 70%. Moreover, the interaction among citrulline, PAH, and probenecid uptakes via rat Oat1 suggested that there are multiple functional sites on Oat1 and that the citrulline site may be distinct from the PAH and probenecid site. Thus, OAT1/Oat1 appear one of a major contributors to renal basolateral uptake of citrulline, and impaired activities of these transporters may contribute substantially to the increase of plasma citrulline in renal failure. Accordingly, citrulline may be useful for diagnosis of kidney function as creatinine is.