Genetic manipulation of Entamoeba histolytica is limited by the inability to express foreign genes at high levels. We tested whether sequences from the E. histolytica rDNA episome, present in 200 copies per cell, could act to stabilize the episomal transfection vector pTCV1. Ligation of the rDNA transcription unit, or sequences downstream of the rDNA transcription unit, increased pTCV1 copy number and stability and conferred additional zones of DNA replication. Sequences upstream of the rDNA transcription unit dramatically destabilized pTCV1. These experiments give additional insights into the mechanism of DNA replication and provide for E. histolytica a set of transfection vectors with unique properties.