Entamoeba histolytica is a phagocytic cell with numerous vesicles of different sizes and shapes but without a well-defined Golgi apparatus. Despite this, genes implied in membrane trafficking have been identified in the genome of this parasite. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein has been shown to play an important role in vesicle fusion in other eukaryotic cells. In this report, we investigated the NSF homologue gene from a pathogenic E. histolytica, characterized its protein product and two of its activities, ATPase and in vitro intra-Golgi transport. The finding of an active NSF protein in E. histolytica indicates that a simple or primordial Golgi apparatus probably exists in this microorganism, as has been proposed by others.