Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2x10(-3)in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites.