Different nutrient bases, fibrinogen or heated plasma preparations and plasminogen sources were compared in tests for staphylokinase production by Staphylococcus aureus and other staphylococcal species. Media containing a nutrient base, bovine fibrin and dog plasma or serum proved to be more sensitive than the other substrates or combinations of substrates tested. Comparative testing on plates wih and without dog plasminogen or with plasmin inhibitors, was found to be necessary for the differentiation of staphylokinase from protease effects. Staphylokinases were produced by S. aureus, S. hyicus, S. simulans, S. sciuri and S. xylosus strains.