Rationale: Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT); capable of clearing pleural adhesions in this model. Methods: Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg) and activities of free urokinase (uPA), plasminogen activator inhibitor 1 (PAI-1) and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. Results: The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t_1/2^β=40±10 min) was dose-independent and 6.7-fold slower than in blood. Up to 260±70 nM of αM/uPA formed in vivo (k_ass=580±60 M^-1s^-1). αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Conclusion: Intrapleural scuPA processing differs from that in the bloodstream and includes (i) dose-dependent control of scuPA activation by endogenous active PAI-1; (ii) two-step inactivation of free uPA with simultaneous formation of αM/uPA; (iii) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.