A highly purified sarcolemma fraction from rat heart consisted of closed inside-out oriented vesicles and possessed high activities of Na+, K+-ATPase, adenylate cyclase and creatine kinase. Contaminations of sarcolemmal preparation by other membranous fractions were practically absent. This sarcolemmal fraction contained protein kinase tightly bound to the membrane. Substrates of the phosphorylation reaction catalyzed by this protein kinase were either endogenous sarcolemmal protein (proteins) with MW 11,500 or exogenous protein-histone, type II. Phosphorylation of the endogenous but not of the exogenous substrate was completely independent of cAMP. A kinetic analysis of the sarcolemmal protein kinase reaction with Mg [.gamma.-32P]ATP and histone as substrates revealed that the kinetic mechanism of this reaction conforms to a sequential quasi-equilibrium Bi-Bi type. The enzyme is characterized by the following kinetic parameters: Km (Mg-ATP) = 12.1 .mu.M; Km (histone) = 0.47 mg/ml; Ki (Mg-ADP) = 15.6 .mu.M. A comparison of experimental results to literary data suggests that the sarcolemmal enzyme is virtually soluble protein kinase tightly bound to the sarcolemma.