The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the beta-subunit, on functional expression of the Na(+)-K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific-[3H]-ouabain binding, ouabain-sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4-6-day-old skeletal myotubes in the presence of TM (1 microgram/ml) for 21-24 hr reduced the number of Na(+)-K+ pumps to 60-90% of control. Na(+)-K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10 degrees C and then re-warming to 37 degrees C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10(-7) M for 24 hr), which blocks voltage-dependent Na+ channels. TM produced significantly greater decreases in ouabain-binding and Em than did TTX, findings that indicate that reduced Na(+)-K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrate that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na(+)-K+ pump beta-subunit is required for full physiological expression of pump activity in skeletal muscle.