Calcium ions (Ca(2+)) play a crucial role in many key physiological processes; thus, the maintenance of Ca(2+) homeostasis is of primary importance. Na(+)/Ca(2+) exchangers (NCXs) play an important role in Ca(2+) homeostasis in animal excitable cells. Bioinformatic analysis of the Arabidopsis genome suggested the existence of a putative NCX gene, Arabidopsis NCX-like (AtNCL), encoding a protein with an NCX-like structure and different from Ca(2+)/H(+) exchangers and Na(+)/H(+) exchangers previously identified in plant. AtNCL was identified to localize in the Arabidopsis cell membrane fraction, have the ability of binding Ca(2+), and possess NCX-like activity in a heterologous expression system of cultured mammalian CHO-K1 cells. AtNCL is broadly expressed in Arabidopsis, and abiotic stresses stimulated its transcript expression. Loss-of-function atncl mutants were less sensitive to salt stress than wild-type or AtNCL transgenic overexpression lines. In addition, the total calcium content in whole atncl mutant seedlings was higher than that in wild type by atomic absorption spectroscopy. The level of free Ca(2+) in the cytosol and Ca(2+) flux at the root tips of atncl mutant plants, as detected using transgenic aequorin and a scanning ion-selective electrode, required a longer recovery time following NaCl stress compared with that in wild type. All of these data suggest that AtNCL encodes a Na(+)/Ca(2+) exchanger-like protein that participates in the maintenance of Ca(2+) homeostasis in Arabidopsis. AtNCL may represent a new type of Ca(2+) transporter in higher plants.