To study mechanisms of K+ transport in peripheral nerve, uptake of rubidium (Rb+), a K+ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K+ Ringer's solutions containing Rb+ (1-20 mM) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb+ (Rb+(o)) and exchanging it for internal K+. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1-10 mM Rb+(o) concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb+ concentrations, which suggests involvement of a nonenzymatic process. At 20 mM Rb+(o), a differential stimulatory response was observed; i.e., axoplasmic Rb+ uptake velocities increased more than fivefold relative to the 10 mM rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb+(o) uptake rate into axons and glia was completely inhibited by ouabain (2-4 mM) exposure or incubation at 4 degrees C. These results suggest that Rb+ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na+,K+-ATPase activity and implicate the presence of axonal- and glial-specific Na+ pump isozymes.