Matrix metalloproteinase 9 (MMP-9) belongs to a family of zinc-dependent endopeptidases. As a consequence of its ability to cleave structural extracellular matrix molecules, mammalian MMP-9 is associated with vital inflammatory processes such as leucocyte migration and tissue remodelling and regeneration. Interestingly, MMP-9 genes have been identified in fish, but functional data are still limited and focus on the involvement of MMP-9 in embryonic development, reproduction and post-mortem tenderization. Here, we describe the involvement of MMP-9 in the innate immunity of carp. In carp, MMP-9 was most notably expressed in classical fish immune organs and in peritoneal and peripheral blood leucocytes, indicating a role of MMP-9 in immune responses. In our well-characterized zymosan-induced peritonitis model for carp, we analysed expression of the MMP-9 gene and the gelatinolytic levels of both pro- and activated forms of MMP-9. The biphasic profile of MMP-9 mRNA expression indicated involvement during the initial phase of inflammation and during the later phase of tissue remodelling. Also, in vitro stimulation of carp phagocytes with lipopolysaccharide or concanavalin A increased MMP-9 gene expression, with a peak at 24 hr. The increase of MMP-9 mRNA correlated with the peak of MMP-9 gelatinolytic level in culture supernatants. These results provide evidence for an evolutionarily conserved and relevant role of MMP-9 in the innate immune response.