We recently described a method for the purification of a protein kinase related to pp60src from Rous sarcoma virus-induced rat tumors (Blithe, D. L., Richert, N. D., and Pastan, I. H. (1982) J. Biol. Chem. 257, 7135-7142). In this report, we describe some of the properties of the 7200-fold purified enzyme. The purified kinase phosphorylates casein, vinculin, actin, and histone H2B, but not bovine serum albumin or ovalbumin. Protein substrates are phosphorylated exclusively on tyrosine residues. Casein was used as a substrate for more detailed analysis. The phosphorylation reaction proceeds at a linear rate for at least 40 min at 22 degrees C. Maximum enzyme activity occurs at pH 6.5 to pH 6.8 and requires the presence of either Mg2+ (5 to 10 mM) or Mn2+ (1 to 5 mM). The Km for ATP is 30 microM and the Vmax 0.03 mumol/min/mg using 0.4 mg/ml of casein as a substrate. The enzyme utilizes ATP or dATP, but not GTP as a phosphate donor in the reaction. The enzyme is inhibited by adenosine and deoxyadenosine and by their corresponding mono- and diphosphates. No inhibition of enzyme activity is observed with adenine, GTP, UTP, TTP, or CTP. The enzyme is very sensitive to increased ionic strength. Addition of 0.1 M KCl, 0.1 M NaCl, 50 mM KPO4, or 50 mM NaPO4 inhibited casein phosphorylation by 90 to 95%. Analysis of the products of the phosphorylation reaction by thin layer chromatography revealed that the src kinase phosphorylates glycerol in addition to casein or the enzyme itself.