The phosphorylation of myofibrillar proteins was studied in rat hearts perfused with [32P]orthophosphate under the influence of negative (M&‘, Cd2’, Pb2’, low Ca’+) and positive (isoproterenol, high Ca’+) inotropic agents. The following physiological parameters were recorded: active tension, spontaneous heart rate, perfusion flow rate, and electrocardiogram. Of these parameters, only the active tension was found to be correlated with protein phosphorylation. The incorporation of [32P]phosphate into the 19,000-dalton light chain of myosin was correlated with both negative and positive inotropy. The incorporation of [32P]phosphate into a 29,000dalton protein with similar electrophoretic mobility to the inhibitory subunit of troponin was correlated with positive inotropy only. A specific Ca’+-sensitive phosphorylation of the 48,000-dalton protein zone was detected. This myofibrillar protein comigrated with cardiac tropomyosin. The phosphorylation of the other myofibrillar proteins was virtually unaffected by the inotropic agents studied. The results support the contention that changes in the phosphorylation of the 19,000-dalton light chain are associated with parallel changes in the contractile state of the myocardium. The present findings and those of previous investigators in conjunction with recent reports on phosphorylation of sarcolemma and sarcoplasmic reticulum proteins (Walsh, D. A., Clippinger, M. S., Sivaramakrishnan, S., and McCullough, T. E. (1979) Biochemistry 18, 871-877; Lindemann, J. P., Jones, L. R., Besch, H. R., Jr., and Watanabe, A. M. (1978) Circulation 58(2), 21) induced by /3-adrenergic stimulation in intact hearts suggest that the regulation of positive inotropy in the heart is through a multiple protein phosphorylation mechanism.