We have constructed a plasmid vector system into which a segment of the Trypanosoma cruzi gene encoding the spliced leader (SL) has been inserted to drive expression of a downstream gene encoding bacterial chloramphenicol acetyltransferase (CAT). We used this construct to establish conditions that permit reproducible transfection of cultured T. cruzi epimastigotes where transfection was mediated by electroporation and measured by assaying expression of CAT. CAT activity was detected only in T. cruzi lysates from cells transfected with constructs containing a properly oriented SL gene; constructs in which the gene was inserted in reverse orientation did not express CAT. The optimal electric field strength, cell density and DNA concentration for efficient transfection were established. This and similar systems will permit genetic dissection of this parasite.