There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to beta(1)-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following beta(1)-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in beta(1)-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca(2+) tension and Ca(2+)-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca(2+)-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca(2+) sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to beta(1)-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.