Ca2+ levels in plants, fungi, and bacteria are controlled in part by H+/Ca2+ exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H+/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca2+ transport. CAX1 has a much higher capacity for Ca 2+ transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca 2+ transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca2+ transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H+/Ca 2+ exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca2+ transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca2+ specificity.