Neutrophil infiltration during zymosan peritonitis depends on matrix metalloproteinase-9 (MMP-9) activity as it is impaired both in MMP-9(-/-) and gelatinase inhibitor-treated animals. The producer cells of MMP-9 and their relative contribution are not known. The aim of this study was to identify and compare the cellular sources, timing and intensity of MMP-9 induction by zymosan in the murine peritoneal cavity. We detected MMP-9 mRNA in unstimulated peritoneal leukocytes and its levels increased after zymosan administration. To detect MMP-9 by flow cytometry, we selected and compared two specific monoclonal antibodies. We show that MMP-9 protein was absent in control peritoneal macrophages, whereas already at 30min of peritonitis almost all macrophages were producing the enzyme. Conversely, MMP-9 was constitutively present in unstimulated mast cells. Macrophages turned out to be prevalent MMP-9 producers in the early phase of peritonitis. During later stages macrophages kept the high expression of MMP-9 for at least 6h of inflammation. In contrast, the early phase expression of MMP-9 by neutrophils was limited albeit the highest percentage of MMP-9(+) neutrophils was observed at 2h but absolute numbers of the MMP-9 carrying neutrophils were low at that time. In contrast, during the late phase of peritonitis neutrophils became major producers of MMP-9 as they numerously infiltrated peritoneum. In conclusion, the study reports detection of MMP-9 at the single-cell level during peritonitis, demonstrates unexpectedly fast MMP-9 expression in macrophages and reveals quantitatively phase-specific contribution of mast cells, macrophages and neutrophils.