Invasive intestinal amebiasis, caused by Entamoeba histolytica, is initiated with attachment of trophozoites to the colonic mucous layer, mucous disruption and/or depletion, and adherence to and cytolysis of host epithelial and inflammatory cells. A current working model of intestinal amebiasis suggests that the microenvironment of the host intestine, particularly intestinal mucins and the bacterial biofilm, may influence the behavior of pathogenic amebae. The invasive phenotype is dependent on expression of a number of virulence factors of which cysteine proteases provide the most convenient experimental probe because their activity is readily monitored. In the present study, we examined the interaction of E. histolytica with GalNAc, mucin, different epithelial cell lines and bacteria both by biochemical assays of protease release and transcriptional profiling using a previously validated genomic microarray. A significant down-regulation of released cysteine protease activity was observed when amebic trophozoites were grown with GalNAc, specific colonic cell lines and bacteria. Transcriptional profiling during GalNAc interaction revealed enhanced expression of the 170-kDa Gal/GalNAc lectin. Decreased protease activity during GalNAc interaction and enhanced expression of the Gal/GalNAc lectin gene are consistent with a program of commensal infection and mucus coat colonization mediated by the lectin. The down-regulation of cysteine protease activity following interaction with a colonic epithelial cell line parallels the presence of secretory mucin having a complex carbohydrate structure rich in Gal and GalNAc. In contrast, interaction of E. histolytica trophozoites with stomach porcine mucin enhanced cysteine protease (EhCP1 and EhCP2) secretion 3-fold. This suggests the specific composition of mucins may affect the Entamoeba phenotype. Transcriptional profiling revealed interaction of Entamoeba with intestinal bacteria induced protein kinase, ABC transporter, Rab family GTPase and hsp 90 gene expression. The enhanced expression of this gene cluster is consistent with enhanced phagocytosis of E. histolytica during interaction with bacteria.