Abstract Matrix metalloproteinases (MMPs) have been implicated in the cleavage of several proinflammatory chemokines, thereby modulating their function and having an impact on the inflammatory process. However, in vivo evidence of such a role remains limited. In this study, we use IL-1β–induced peritonitis as a model for an acute immune response, which is initiated by neutrophil influx followed by macrophage infiltration within a few hours of IL-1β injection into the peritoneal cavity. Using single and double knockout mice for MMP-2 and MMP-9, we show that MMP-2 and MMP-9 act synergistically mainly at the initial step of neutrophil recruitment into the peritoneal cavity. The use of bone marrow chimeric mice revealed the cellular sources of MMP-2 and MMP-9 to be distinct, with resident cells being the source of the former and infiltrating leukocytes the source of the latter. We show that the omentum is the main site of neutrophil entry into the peritoneal cavity, where MMP-2 and MMP-9 act synergistically to potentiate the action of CXCL5 (ENA-78/ LIX), thereby, promoting neutrophil migration into the peritoneal cavity. To our knowledge, this is the first in vivo demonstration of MMP-2 and MMP-9 processing of a chemokine that has been directly correlated with an enhanced chemoattracting function.