Entamoeba histolytica, a protozoan parasite, is an important human pathogen. Diseases caused by E. histolytica include dysentery and liver abscesses, and the organism is a leading parasitic cause of death on a global scale. Regulation of gene expression is a key factor that enables the parasite to adapt to the host environment during tissue invasion and to convert between life stages to propagate disease outside the host. One mechanism of gene regulation in Entamoeba is a robust and complex endogenous RNAi pathway. Over the last several years we have made important observations about this pathway including that amebic small RNAs have highly unusual 5'-polyP termini and associate with amebic Argonaute 2-2 protein to mediate transcriptional gene silencing. Additionally, we identified that Entamoeba contains three Argonaute proteins with different cellular localizations and apparently non-redundant functions. We have also used information on the amebic RNAi pathway to develop a novel and robust gene-silencing tool in Entamoeba based on use of a “trigger” gene. Trigger gene silencing is highly robust and stable and is mediated by repressive histone modifications on the trigger-silenced loci. Our goal is to focus on aspects of RNAi in ameba that are either unique compared to other systems or which intersect with important aspects of parasite biology. Thus, we will (i) determine the molecular mechanisms of small RNA “trigger” mediated gene silencing, and (ii) characterize the components of the Argonaute RNA induced silencing complexes. These data will improve our understanding of the molecular mechanisms of RNAi in Entamoeba. Our work is at the intersection of the basic cellular process of RNA-interference and amebic biology. Data that emerge will contribute to both understanding a basic process in amebic biology and to expanding the knowledge about the fundamental process of RNAi.