The role of the electrogenic Na+-K+ pump in the determination of the level of the resting membrane potential in cultured chick limb muscle was investigated. Transmembrane resting potential and ouabain-sensitive 86Rb-uptake were measured in myotubes at different ages in culture from 2 to 10 days in vitro. Inhibition of the Na+-K+ pump with ouabain prevented the developmental increase in membrane potential which normally follows fusion of myotubes (day 2-3). In mature myotubes, ouabain caused a dose-related decrease in both membrane potential and 86Rb-uptake, with values for EC50 and maximal effect being nearly the same on both variables. The decrease in membrane potential by ouabain, up to 20 mV maximum, occurred within 2-5 sec and was not accompanied by detectable changes in input resistance. Membrane potential was also reduced by a decrease in temperature of the recording medium and removal of extracellular K+, both of which reduce Na+-K+ pump activity. We also found that the relation between membrane potential and extracellular K+ concentration was completely attenuated by ouabain in the physiological range (2-10 mM). We conclude that the electrogenic Na+-K+ pump plays an important role in the determination of the resting membrane potential of chick myotubes and that regulation of its level is not entirely explained by the diffusion potential hypothesis.