The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite-host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.