The importance of RLC phosphorylation in enhancing cardiac myofibrillar contraction is well-known. In anesthetized mice the extent of RLC phosphorylation (45%) was not changed by changes in sympathetic tone with prolonged infusion of the beta-agonist dobutamine or treatment with the beta-blocker propranolol. The goal of this study was to determine if the constitutive RLC phosphorylation in vivo was limited to half of the RLC due to: a) negative cooperativity for phosphorylation of two heads in myosin; or b) due to a steric constraint in myofibrils blocking access of soluble cardiac myosin light chain kinase (cMLCK) to RLC. We measured the kinetic properties of RLC phosphorylation in native myosin filaments and myofibrils. Results showed that RLC was phosphorylated by a pseudo-first order rate in both preparations with maximal phosphorylation over 90%. Thus, RLC for each head in myosin was readily available for phosphorylation. Pacing trabeculae at 1.5 Hz for 30 minutes increased RLC phosphorylation from 20±1 % to 43±3 %. Consistent with biochemical results, RLC phosphorylation increased to 91±3 % when myosin light chain phosphatase activity was inhibited with calyculin A while pacing. Together, these results exclude negative cooperativity and steric blocking as mechanisms limiting RLC phosphorylation. We determined that the heart has a high cMLCK content (2.4±0.1 µM) compared to the MLCK present in fast skeletal muscle (0.5±0.03 µM), but similar to the MLCK content in smooth muscle (3.4±0.2 µM). However cMLCK has a low specific activity compared to the other MLCKs. In conclusion, the extent of RLC phosphorylation in a normally beating heart is limited by cMLCK with its low activity in balance with low myosin light chain phosphatase activity. RLC phosphorylation is insensitive to sympathetic activation or inhibition in vivo.