A sensitive in situ assay for the plasminogen activator of transformed cells is described; it uses the fluorogenic molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-(N'-4-carboxylphenyl)thioureidospiro[isobenz ofuran-1(3H),9'-[9H]xanthen]-3-one. This fluorescein derivative is an excellent active-site titrant of the esterase activity of plasmin. When transformed cells are incubated with purified plasminogen and the resulting plasmin is titrated with the fluorogenic substrate, the amount of plasmin formed is linearly proportional to time and cell number. The assay is sensitive enough to detect quantitatively the plasminogen activator activity of as few as 250 transformed cells. This substrate should be useful in studying quantitatively the correlation between increased levels of plasminogen activator activity and cellular transformation and as a general active site titrant of serine proteases.