Technical Abstract: The Arabidopsis ("Arabidopsis thaliana") cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant "CCXs" have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na(+), K(+), and Mn(2+) transport. We also report high-capacity uptake of (86)Rb(+) in tonoplast-enriched vesicles from yeast expressing "AtCCX3". Cation competition studies showed inhibition of (86)Rb(+) uptake in "AtCCX3" cells by excess Na(+), K(+), and Mn(2+). Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, "AtCCX3" is primarily expressed in flowers, while "AtCCX4" is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of "AtCCX3" increased in plants treated with NaCl, KCl, and MnCl[2]. Insertional mutant lines of "AtCCX3" and "AtCCX4" displayed no apparent growth defects; however, overexpression of "AtCCX3" caused increased Na(+) accumulation and increased (86)Rb(+) transport. Uptake of (86)Rb(+) increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of "AtCCX3" in tobacco ("Nicotiana tabacum") produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H(+)-dependent K(+) transporter with apparent Na(+) and Mn(2+) transport properties distinct from those of previously characterized plant transporters.