We hypothesized that 20S proteasome is present and functional in the extracellular alveolar space in humans. Proteasomal activity was measured in bronchoalveolar lavage (BAL) supernatant from eight humans using specific proteasomal fluorogenic substrates and I(125)-albumin with and without specific proteasome inhibitors. Furthermore, gelfiltration, Western blot technique, and mass spectrometry were applied for proteasome characterization. All proteasomal fluorogenic substrates were hydrolyzed by BAL supernatant, with hydrolysis inhibited by epoxomicin (P = 0.024) and other proteasome inhibitors as well. E64, a lysosomal inhibitor, did not inhibit enzyme activity. The majority of proteolytic activity was detected in BAL supernatant rather than in the cell pellet. No correlation was found between proteasomal hydrolysis in BAL supernatant and lactate dehydrogenase activity, the total cell count in the cell pellet, and the fraction of avital cells in the cell pellet, ruling out cell lysis as a major source of proteasomal activity. Gelfiltration revealed hydrolyzing activity in the supernatant at 660 kDa and proteasome core proteins after analysis by ESI-QqTOF mass spectrometry. Furthermore, Western blots using a polyclonal antibody against proteasomal alpha-/beta-subunits detected proteasomal proteins in the typical 20- to 30-kDa range in BAL supernatant. Incubation of BAL supernatant with I(125)-albumin showed a high mean cleavage rate (101.8 microg/ml x h lavage +/- 46 SD) that was inhibited by epoxomicin (P = 0.013) and was ATP and ubiquitin independent. We identified for the first time extracellular, biologically active, ATP- and ubiquitin-independent 20S proteasome in the human alveolar space, with a high albumin cleavage rate. Possibly, the proteasome assists in maintenance of a low intra-alveolar oncotic pressure and/or alveolar protein degradation.