Background: Myosin cross-bridges containing phosphorylated and dephosphorylated regulatory light chain may be different. Results: Relaxed cross-bridges, but not active ones, are better ordered in muscle containing dephosphorylated RLC than phosphorylated RLC; they both rotate equally slowly. During contraction phosphorylated cross-bridges rotate faster. Conclusion: Both types of cross-bridges are functionally different. Significance: Phosphorylation of skeletal myosin RLC plays a role in regulation of skeletal muscle contraction.