In a search for a probe which would report its proteolysis to thrombin, the human blood coagulation zymogen prothrombin was covalently labeled with fluorescein. Fluorescein isothiocyanate (FITC) and dichlorotriazinylaminofluorescein (DCTAF) both introduced approximately 1 molecule of dye, but labeling occurred at different locations, as FITC had no effect on clotting activity whereas DCTAF caused 95% inactivation. At pH 9.0 DCTAF, but not FITC, could induce labeling up to 4 mol/mol. All derivatives were activated normally by prothrombinase (the activating complex of Factor Xa, Factor V(a), Ca2+ and phospholipids), as indicated by the pattern of bands on SDS gel electrophoresis and an unaltered yield of activity toward a chromogenic substrate for thrombin. Upon undergoing this limited proteolysis, the most heavily labeled derivative showed a 40% increase in fluorescence of the fluorescein at 520 nm (lambda ex 480 nm). In contrast, the fluorescence of lightly labeled forms was more intense but increased by only 0-5% upon activation. The data suggest that the lower fluorescence of the most labeled form is due to an intramolecular quenching effect between the dye molecules on individual polypeptide chains that is partly relieved when activation occurs.