Degradation of the extracellular matrix during cancer invasion is accomplished by the concerted action of several proteolytic enzymes, including matrix metalloproteinases (MMPs). We have studied the immunohistochemical localization of one of these enzymes, 92-kDa type IV collagenase (MMP-9), in short-term fixed specimens of 19 colon adenocarcinomas and 2 biopsies of adjacent normal colon. Staining was confined to neutrophils and macrophages, as identified by double staining. All neutrophils were positive in all cases. Some positively stained tumor-infiltrating macrophages were seen in 6 (32%) of the tumors, located adjacent to invasive tumor glands. No cancer cells were stained in any of the cases. In normal colon tissue, staining was only seen of scattered neutrophils in vessels and of macrophages in Peyer's patches. Routinely processed specimens from 7 of the 19 carcinomas were analyzed by in situ hybridization. In agreement with previous results, a MMP-9 mRNA signal was in all cases seen in a subpopulation of tissue macrophages surrounding invasive tumor glands, while no MMP-9 mRNA was detected in any other cell types, including neutrophils and cancer cells. Our results indicate that in this type of cancer all neutrophils contain MMP-9, which has been produced before they infiltrate the tumors; that a subpopulation of the tumor-infiltrating macrophages most likely in all cases produces MMP-9 but that the content of this protein is low due to a rapid turnover and that malignant epithelial cells do not produce or contain detectable amounts of MMP-9. These findings extend previous results indicating that stromal cells are actively involved in the generation and regulation of extracellular proteolysis during cancer invasion.