Increasing the K+ from 5.4 mM to 12 mM in the culture medium of developing chick myotubes causes an immediate cessation of spontaneous contractions and leads to an inhibition of myosin accumulation. The synthesis of myosin continues at the same rate in 12 mM K+ as in 5.4 mM K+ as measured by [3H]leucine incorporation into myosin corrected for differences in pool specific activity. Total protein synthesis and total protein accumulation are unaffected by growth in 12 mM K+. In addition, growth in 12 mM K+ did not alter the type of myosin heavy-chain isoform expression nor did it alter the pattern of myosin light-chain synthesis. However, the rate of myosin turnover increased threefold in cultures grown in 12 mM K+ compared to cultures grown in 5.4 mM K+, while total protein turnover was only marginally increased. We conclude that suppressed electrical or contractile activity of myotubes leads to an increased rate of myofibrillar protein turnover and that spontaneous mechanical and or electrical activity is required for continued myotube maturation in culture.