High molecular weight ADP ribosylation factor GDP-GTP exchange factors (ARF-GEF) play an essential role in the formation of COP I coated transport vesicles and are characterized by a structurally and functionally conserved sec 7 domain. The genome of the malaria parasite Plasmodium falciparum encodes a single ARF-GEF that contains an unusual sec 7 domain. In comparison to the sec 7 domain of other eukaryotes, the plasmodial sec 7 domain is characterized by an insertion sequence of 146 amino acids that disrupt helices essential for the GDP-GTP exchange activity of the protein. In a previous study we have shown a correlation between a methionine to isoleucine exchange in helix H of the sec 7 domain and resistance to brefeldin A in a parasite line generated by drug selection. Here we have transfected brefeldin A sensitive parasites with plasmid constructs containing the sec 7 domain of the resistant line either with or without the insertion sequence. Transfection with sec 7 sequences including the insertion resulted in brefeldin A resistant parasites in which double cross-over recombination had replaced the endogenous sec 7 sequences with the transgenic sequences. Thus, the point mutation in helix H is sufficient to confer brefeldin A resistance in P. falciparum. Transfections using constructs lacking the insertion did not result in resistant parasites. Gene replacement by targeted double cross-over recombination is a rare event in P. falciparum. This approach has taken advantage of the fact that the successful integration of the transgene results in a drug selectable phenotype. We anticipate that the strategy described here will be useful for the identification of mutations within target genes that have the potential to confer increased drug resistance.