In these studies the fibrinogen plate medium proved to be not sufficiently specific for the demonstration of staphylokinase (SAK). For this purpose we modified the azocasein-test by the addition of plasminogen. Protease, on the other hand, we measured without plasminogen. This enabled us to differentiate between the 2 enzymes. After precipitation with ZnCl2 from the culture supernatant of Staphylococcus aureus strain, V8, SAK could be prepurified by filtration on ultrogel AcA 44 (tab. 1, fig 1). A more than 100-fold increase in specific SAK-activity (in comparison to that in the culture supernatant) to 34.722 units/mg protein was achieved after 2 X isoelectric focusing between pH 3.5-10.0 and refocusing between pH 5.0-7.0 (fig 2). The partially purified SAK was free of protease, coagulase, beta- and delta-hemolysins. DNase, lipase and phosphatases, but it contained minor amounts of alpha-hemolysin. It revealed only 1 band in the SDS-polyacrylamide-gel-electro-phoresis and 1 precipitin-line in the double immunodiffusion test with an antiserum against the SAK preparation after ultrogelfiltration.