In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H+ antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca2+ into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn2+ transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn2+ specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn2+ or Ca2+ transport, the second class showed a reduction in Ca2+ transport and no measurable Mn2+ transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca2+ transport and a complete loss of Mn2+ transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn2+ specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H+ antiporter, an important family of transporters found in a variety of organisms.