Publisher Summary This chapter describes the phosphorylation of human immune interferon (IFN-γ) with cyclic adenosine monophosphate (cAMP)-dependent protein kinase from bovine heart. The phosphorylation reaction should be useful for the preparation of radioactive IFN-γ for receptor studies. cAMP-dependent protein kinase catalyzes the transfer of the γ-phosphate of Adenosine-5'-triphosphate (ATP) to serine and/or threonine hydroxyl groups in various protein substrates. IFN-γ can be labeled simply and efficiently with the catalytic subunit of cAMP-dependent protein kinase from bovine heart. Beef heart protein kinase catalyzes the phosphorylation of protamine, arginine-rich histone, and lysine-rich histone at relative rates of 10:5:2. Serum albumin and casein are poor substrates. IFN-γ is a basic protein with high content of arginine and lysine (about 20%). IFN-γ can be used as a substrate for bovine heart protein kinase. The chapter also discusses the in vitro phosphorylation of IFN-γ to high specific radioactivity with [γ-32P]ATP in the absence of unlabeled ATP. This radioactive IFN-γ with full biological activity is suitable for receptor studies. Preliminary data indicate that the 32P-labeled IFN-γ binds specifically to IFN-γ receptors of human cell lines.