The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [(35)S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time- and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.