Abstract: Phosphorylated plasminogen activator, such as phosphorylated pro-urokinase (pro-u-PA), which is substantially free from unphosphorylated plasminogen activator, may be obtained by phosphorylating unphosphorylated plasminogen activator with a phosphorylating enzyme or by separating phosphorylated plasminogen activator from a mixture of phosphorylated plasminogen activator and unphosphorylated plasminogen activator. Phosphorylated pro-u-PA, which is substantially free from unphosphorylated pro-u-PA, is converted by plasmin into phosphorylated u-PA. The phosphorylated plasminogen activators such as phosphorylated pro-u-PA, u-PA and t-PA are useful as thrombolytic agents.