Recent observations suggest the presence of 20S proteasomes (20S) in the lung epithelial lining fluid. However, the physiological relevance of 20S in the alveolar space and possible contribution to disease processes are unknown. Thus, we evaluated whether extracellular proteasomes could have a pathophysiological role in the injured lung using a rat model of lung contusion (LC). Bronchoalveolar lavage fluids (BALF) were obtained at various time points for up to 168 h after LC or sham procedure. Enzyme activities, ELISA and Western blots indicated enzymatically active 20S, the 19S subunit Rpt5 and ubiquitin in BALF. 20S and ubiquitin increased significantly after LC, peaked at 24 h and normalized within 168 h. Mg(2+)/ATP-dependent peptidase activities were detectable 6-24 h after LC. BALF after LC also contained ubiquitin-protein-ligase activity. Addition of Mg(2+)/ATP to BALF after LC led to significant proteolysis and could be prevented with epoxomicin and EDTA. These data suggest for the first time that the Mg(2+)/ATP-dependent 26S proteasome complex exists outside the cell, is released into the lung epithelial lining fluid after LC and contributes to the proteolysis of the bulk of protein in the alveolar space of the injured lung. We infer that proteasome complexes may have a pathophysiological role during lung edema clearance.