Plasmin is the key enzyme of fibrinolysis. Plasmin-alpha2-antiplasmin (PAP) complexes are biomarkers of fibrinolysis activation. The purpose of our investigation was to evaluate the activity of fibrinolysis in normal human eyes, that is in eyes without blood-retina barrier breakdown (BRB), which has not been investigated so far. Twenty-two vitreal samples were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy for macular pucker removal, macular hole closure or vitreal floater removal from the central vitreous body. These samples were immediately stabilized with human albumin (2.5% final conc.) and arginine (1.25 mmol/l final conc.) and subsequently frozen. Plasminogen was functionally determined in an ultra-sensitive pNA reaction after activation with streptokinase (100% = functional plasminogen in pooled normal citrated plasma). PAP concentrations were measured by enzyme immune assay (EIA). Intravitreal functional plasminogen exhibited to be 1 ± 0.65% (range: 0.2-2.49%). PAP concentrations ranged at levels of 14 ± 9ng/ml (range: 2-33 ng/ml). Pearson's correlation quotient between functional plasminogen and PAP revealed to be r equal to -0.27 (P = 0.221). No adverse events or serious side effects occurred. Sampling vitreous fluid at the beginning of a standard 23-gauge three-port pars plana vitrectomy is a well tolerated procedure. A strict stabilization procedure for extracted vitreous specimen is necessary to obtain activities and concentrations that are close to the true intraocular value. There is a basal intraocular fibrinolysis, a possible target for intravitreal pharmacological therapy.