Secondary peritonitis is an important indication for surgical intensive care admissions, and it is associated with high morbidity and mortality. Collagenase 2/matrix metalloproteinase (MMP) 8 is a tissue matrix-degrading enzyme that is released from leukocytes upon inflammatory stimuli and may thus contribute to peritonitis-associated organ damage. We studied the levels and activity of MMP-8 in the peritoneal fluid of 15 critically ill patients with secondary peritonitis. The MMP-8 levels were measured from the patients' peritoneal fluid, serum, and urine, and from the serum and urine of 10 healthy controls by immunofluorometric assay. Median MMP-8 level in peritoneal fluid supernatant was 1,317 μg/L (interquartile range [IQR]) (1,254-1,359 μg/L) being significantly higher than in the sera of the patients (P = 0.008). Molecular forms and isoform distribution of MMP-8, MMP-1, and MMP-13 in peritoneal fluid, assessed by Western immunoblotting, revealed that the neutrophil-type MMP-8 was the major collagenase species in peritoneal fluid, and it was partially in an activated form. Catalytically competent, active MMP-8 produced the characteristic cleavage products from intact human type I collagen. The serum levels of MMP-8 were higher in the patients, 49 μg/L (IQR, 23-214 μg/L), than in the controls, 11 μg/L (IQR, 8-24 μg/L) (P < 0.01). The MMP-8 levels in the urine were higher in the patients, 0.27 μg/L (IQR, 0.04-1.89 μg/L), than in the controls, 0.03 μg/L (IQR, 0.0-0.05 μg/L) (P = 0.013). Our data demonstrate for the first time that MMP-8 levels are remarkably elevated and in an active and catalytically competent form in the peritoneal fluid samples of patients with secondary peritonitis.