The Ca2+-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p. In yeast and plants, cation/H+ exchangers are important in shaping cytosolic Ca2+ levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca 2+, Mn2+ and Cd2+. Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca2+ transporter and may also function in Cd2+ transport, but cannot transport Mn2+. Furthermore, direct Ca2+ transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn2+ tolerance. Here we demonstrate that this Mn2+ tolerance is independent of the Ca 2+/Mn2+-ATPase Pmr1p and results from Mn 2+-specific vacuolar transport activity of Vcx1-M1p. This Mn 2+ transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn2+ tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.