Abstract Potassium (K+) is the most abundant cation in plants and is required for plant growth. To ensure an adequate supply of K+, plants have multiple mechanisms for uptake and translocation. However, relatively little is known about the physiological role of proteins encoded by a family of 13 genes, named AtKT/KUP, that are involved in K+ transport and translocation. To begin to understand where and under what conditions these transporters function, we used reverse transcription-PCR to determine the spatial and temporal expression patterns of each AtKT/KUP gene across a range of organs and tested whether selected AtKT/KUP cDNAs function as K+ transporters in Escherichia coli. Many AtKT/KUPs were expressed in roots, leaves, siliques, and flowers of plants grown under K+-sufficient conditions (1.75 mm KCl) in hydroponic culture. AtHAK5 was the only gene in this family that was up-regulated upon K+ deprivation and rapidly down-regulated with resupply of K+. Ten AtKT/KUPs were expressed in root hairs, but only five were expressed in root tip cells. This suggests an important role for root hairs in K+ uptake. The growth and rubidium (Rb+) uptake of two root hair mutants, trh1-1 (tiny root hairs) and rhd6 (root hair defective), were studied to determine the contribution of root hairs to whole-plant K+ status. Whole-plant biomass decreased in the root hair mutants only when K+ concentrations were low; Rb+ (used as a tracer for K+) uptake rates were lower in the mutants at all Rb+ concentrations. Seven genes encoding AtKUP transporters were expressed in E. coli (AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5), and their K+ transport function was demonstrated.