Mass drug administration, a key malaria control strategy, has been thwarted by the development of drug resistance to nearly all classes of antimalarials in use. Antifolate drugs are antimalarial compounds that target and inhibit the folate pathway necessary for pyrimidine synthesis in the blood stage parasite. Discovering and understanding the evolution of resistance is essential to developing novel treatment regimes and avoiding future treatment failure. Previous studies of P. falciparum malaria parasites derived from the field isolates suggest an association between gch1 copy number variations (CNVs) and resistance to the antimalarial drug pyrimethamine (PYR). However, the actual functional role of this CNV on drug sensitivity and parasite fitness has not been determined. Using the parents and progeny of the genetic cross between clonal parent lines, HB3 and Dd2, we identified a positive correlation between gch1 copy number (CN) and PYR IC50 values and confirmed the phenotypic effect of the gch1 CNV by amplifying the CN in HB3 parasites (a two-fold increase) using the piggyBac transfection system. Our data raise the interesting possibility that the genetic background of resistant parasites, specifically dihydrofolate reductase (DHFR) mutation background, influences the magnitude of effect of gch1 CNV on parasite drug response. Previously, CNVs have been associated with resistance levels in natural parasite populations, however evidence for their direct impact on drug-related traits was lacking. A key outcome of this project was the development of approaches for the continued study of the gch1 CN phenotype. New transfected parasites are already being produced to further explore the role of dhfr background mutations. We have also developed a multiple-reaction monitoring mass- spectroscopy assay to measure changes in flux of the folate biosynthesis pathway caused by the gch1 amplification. This method will enable experiments to determine the mechanism by which gch1 amplification we leads to shifts in drug susceptibility.