Current models of potassium acquisition and cytochemical processes in plants assume that potassium concentrations in the cytosol ([K+]cyt) are maintained homeostatically at approximately 100 mM. Here, we use 42K radiotracer data in the model plant species Hordeum vulgare L. (barley) to show that this assumption is incorrect. Our study reveals that [K+]cyt in root cells of intact barley seedlings is held at a minimum of two physiological set points, coinciding with two fundamentally distinct modes of K+ transport, each of which is characterized by a unique network of fluxes to and from the cytosol, and reflects variations in mechanisms and energetics of K+ transport, cytosolic K+ turnover, flux partitioning, and sensitivity to NH4+. Increased external potassium or ammonium concentrations caused a substantial drop in [K+]cyt, as well as a switch from a transport mode dominated by high-affinity, energy-dependent, influx to a mode dominated by channel-mediated fluxes in both directions across the plasma membrane. Our study provides the first subcellular demonstration of the flexibility, rather than strict homeostasis, of cellular K+ maintenance, and of the dynamic interaction between plant membrane fluxes of the two major nutrient cations K+ and NH4+.