In hospital-acquired pneumonia, extracellular matrix destruction is common and may be caused by excessive activity of matrix metalloproteinases (MMPs). Thirty patients with hospital-acquired pneumonia and 16 control subjects were studied. We evaluated the concentrations of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 in mini-bronchoalveolar lavage fluid (mini-BALF) and blood using zymography and specific immunoassays. In patients with hospital-acquired pneumonia concentrations of MMP-8 and MMP-9 in mini-BALF were increased 10-fold, whereas their specific inhibitor tissue inhibitor of metalloproteinase-1 was not concomitantly increased. In 80% of patients with pneumonia, but in none of the control subjects, the active form of MMP-9 was detected by zymography. Zymography furthermore showed the banding pattern of neutrophil-derived MMP-9, indicating that neutrophils were the main source of MMP-9. Comparison of neutrophils from blood and mini-BALF showed higher basal release of MMPs by pulmonary neutrophils. Stimulation analysis indicated that pulmonary neutrophils were already maximally activated. In patients with detection of potentially pathogenic microorganisms, concentrations of MMPs were fivefold increased compared with patients with negative cultures. Furthermore, MMP-levels were related to clinical severity. These are the first data suggesting that neutrophil-derived MMPs are increased in hospital-acquired pneumonia in association to the detection of causative microorganisms and clinical severity.