In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca2+-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% +/- 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca2+ (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca2+/calmodulin-dependent kinase and a Ca2+/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities. (C) 2016 Elsevier Inc. All rights reserved.