Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2Ac) from bovine kidney resulted in time-dependent inactivation of the protamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2Ac was completely inhibited. Half-maximal inhibition by ATP occurred at about 20 microM. The rate of inactivation of the protamine kinase by PrP2Ac was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at 1 mM. The results strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation.