The plant vacuole is the primary storage site in plant cells for micro and macronutrient ions, and pollutant heavy metal ions. In tobacco, nutrient ions such as K and Cl, and pollutant ions such as Cd are important to product quality. During growth, K and Mn, under certain conditions, can substantially impact growth and yield. All of these ions are known to be primarily stored/sequestered in the vacuole within cells. Therefore, mechanisms for transport of ions across the vacuolar membrane (tonoplast) are important to ion homeostasis, plant stress (drought, salinity, nutrient etc..) responses and pollutant metal accumulation. Most studies of root tonoplast ion transport have used seedlings of oat, corn, or tap roots of red beet. Here we report the isolation of transport-competent vesicles from soil-grown mature tobacco roots. Characterization of Ca and Cd transport in mature tobacco root and seedling root vesicles were compared and found to be similar. Solubilization of vesicles and reconstitution with artificial lipids reduced protein and abolished Ca transport, but retained Cd transport in a manner similar to that observed with oat seedling root tonoplast. These results suggest that monocot and dicot, seedling and mature root tonoplast have similar properties with respect to Ca and Cd transport.