The positively charged fluorescent dyes ethidium (Et⁺) and propidium (Pr²⁺) are widely used as DNA and necrosis markers. Et⁺ is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2) and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT) whereas in rodents, OCT1/2 are involved. In mouse kidney, perfused Et⁺ accumulated predominantly in the S2/S3 segments of the PT, but not Pr²⁺. In cells stably overexpressing human OCTs (hOCTs) Et⁺ uptake was observed with K_m values of 0.8±0.2µM (hOCT1), 1.7±0.5µM (hOCT2) and 2.0±0.5µM (hOCT3), whereas Pr²⁺ was not transported. Accumulation of Et⁺ was inhibited by OCT substrates quinine, 3-methyl-4- phenylpyridinium (MPP⁺), cimetidine and tetraethylammonium (TEA⁺). For hOCT1 and hOCT2, the IC₅₀ values for MPP⁺, TEA⁺, and cimetidine were higher than for inhibition of previously tested substrates. For hOCT2 the inhibition of Et⁺ uptake by MPP⁺ and cimitidine was shown to be competitive. Et⁺ also inhibited transport of 0.1µM [³H]-MPP⁺ by all hOCT isoforms with IC₅₀ values between 0.4 and 1.3µM, and the inhibition of hOCT1 mediated uptake of MPP⁺ by Et⁺ was competitive. In Oct1/2^-/- mice, Et⁺ uptake in the PT was almost abolished. The data demonstrate that Et⁺ is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et⁺ and their strong expression in various organs, strict safety guidelines for Et⁺ handling should be reinforced.