Troponin T kinase was purified from dog heart extracts to apparent homogeneity (specific activity, 0.8-1.1 mumol of P incorporated/mg of protein/min at 30 degrees C). Analysis of the purified enzyme by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed two major protein bands corresponding to Mr = 37,000 and 28,000. The Mr = 28,000 band was found to be phosphorylated after incubation of the enzyme with [32P]ATP.Mg. The molecular weight of the enzyme was calculated to be approximately 125,000 from the results of gel filtration on Sephacryl S-300. It would appear that cardiac troponin T kinase is composed of two 37,000- and two 28,000-dalton subunits. The purified kinase was found to phosphorylate troponin T from cardiac or skeletal muscle even in the presence of other components of the troponin system. Phosvitin and casein were also phosphorylated by the enzyme. The Km for ATP was determined to be 7 microM; free Mg2+ was required for maximal activity. Calcium ions, either in the presence of absence of calmodulin, had only a slight inhibitory effect. Addition of fluoride ions (50 mM) and arginine-rich histone (1 mg/ml) to the reaction mixture resulted in a strong inhibition of the activity. Cyclic nucleotides did not affect the kinase activity.