The role of Na-K ATPase in the determination of resting membrane potential (Em) as a function of extracellular K ion concentration was investigated in cultured rat myotubes. The Em of control myotubes at 37 degrees C varied as a function of (K+)0 with a slope of about 58-60 mV per ten-fold change in (K+)0. Inhibition of the Na-K pump with ouabain or by reduced temperature revealed that this relation consists of two components. One, between (K+)0 of 10 and 100 mM, remains unchanged by alterations in enzyme activity; The second, between (K+)0 of 1 and 10 mM, is related to the amount of Na-K pump activity, the slope decreasing as pump activity decreases. Indeed, with complete inhibition of the Na-K pump, Em does not change over the range of (K+)0 1 to 10 mM. Measurements of 86Rb efflux and input resistance of individual myotubes showed that membrane permeability does not change as (K+)0 increases from 1 to 10 mM but increases as (K+)0 increases further. Monensin, which increases Na ion permeability, increases Em at values of external K+ below 10 mM, and is without effect at higher values of K+ concentration. The effect of monensin is blocked by ouabain. Tetrodotoxin, which blocks voltage-dependent Na+ channels, decreases Em at low (2-10 mM) K+. We conclude that changes in Em as a function of extracellular K+ concentration in the physiological range are not adequately explained by the diffusion potential hypothesis of Em, and that other theories (electrogenic pump, surface-absorption) must be considered.