In order to study factors affecting homologous recombination in P. falciparum, two constructs were prepared containing the Toxoplasma gondii dhfr-ts gene as the selectable marker. In the first construct (pDHD-Tg23), the T. gondii dhfr-ts was flanked by 5' and 3' untranslated flanking regions (UTFRs) from dhfr-ts (chr 4), as well as the 5 ' UTFR from histidine rich protein 3 (hrp3, chr 13). In the second construct (pHSP-Tg23), the T. gondii dhfr-ts gene was flanked by the 5' and 3' UTFRs from heat shock protein 86 (hsp86, chr 7). After transfection of the pyrimethamine-susceptible 3D7 and Haiti 135 strains of P. falciparum by electroporation and selection with pyrimethamine, individual pyrimethamine-resistant clones were selected by limiting dilution. In contrast to the parental 3D7 and Haiti 135 parasites, these pyrimethamine-resistant clones contained both the P. falciparum and T. gondii dhfr-ts genes. Based on chromosomal electrophoresis and hybridization, the P. falciparum dhfr-ts gene was consistently localized to chr 4 in both the parental strains and the transfectants. In contrast, the T. gondii dhfr-ts gene was found on chr 7, and chr 13, after transfection with pHSP-Tg23, and pDHD-Tg23, respectively. Southern blotting and PCR amplification suggest that deletions occurred in the transfected constructs. Two or three copies of the pDHD-Tg23 DNA are present on chromosome 13 (3D7) and 1 copy of the pHSP-Tg23 DNA is on chromosome 7 (Haiti 135) of the pyrimethamine-resistant transfectants. These results suggest that it is possible to direct genes to specific chromosomes in P. falciparum, based on the UTFRs flanking the marker in the construct used for electroporation and selection